Methods for expression and purification of recombinant human growth hormone

ABSTRACT

The present invention relates generally to the production, purification, and isolation of human growth hormone (hGH). More particularly, the invention relates to the production, purification, and isolation of substantially purified hGH comprising non-naturally encoded amino acids and hGH from recombinant host cells or culture medium including, for example, yeast, insect, mammalian and bacterial host cells. The process of the present invention is also useful for purification of hGH linked to polymers or other molecules.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. provisional patent applicationSer. No. 60/638,616 filed Dec. 22, 2004, U.S. provisional patentapplication Ser. No. 60/655,744 filed Feb. 23, 2005, U.S. provisionalpatent application Ser. No. 60/680,977 filed May 13, 2005, and U.S.provisional patent application Ser. No. 60/727,968 filed Oct. 17, 2005,the specifications of which are incorporated herein in their entirety.

FIELD OF THE INVENTION

The present invention relates generally to the production, purification,and isolation of human growth hormone (hGH). More particularly, theinvention relates to the production, purification, and isolation ofsubstantially purified hGH from a recombinant host.

BACKGROUND OF THE INVENTION

The growth hormone (GH) supergene family (Bazan, F. Immunology Today 11:350-354 (1990); Mott, H. R. and Campbell, I. D. Current Opinion inStructural Biology 5: 114-121 (1995); Silvennoinen, O. and Ihle, J. N.(1996) SIGNALING BY THE HEMATOPOIETIC CYTOKINE RECEPTORS) represents aset of proteins with similar structural characteristics. Each member ofthis family of proteins comprises a four helical bundle. While there arestill more members of the family yet to be identified, some members ofthe family include the following: growth hormone, prolactin, placentallactogen, erythropoietin (EPO), thrombopoietin (TPO), interleukin-2(IL-2), IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12 (p35subunit), IL-13, IL-15, oncostatin M, ciliary neurotrophic factor,leukemia inhibitory factor, alpha interferon, beta interferon, gammainterferon, omega interferon, tau interferon, epsilon interferon,granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophagecolony stimulating factor (GM-CSF), macrophage colony stimulating factor(M-CSF) and cardiotrophin-1 (CT-1) (“the GH supergene family”). Membersof the GH supergene family have similar secondary and tertiarystructures, despite the fact that they generally have limited amino acidor DNA sequence identity. The shared structural features allow newmembers of the gene family to be readily identified.

Human growth hormone participates in much of the regulation of normalhuman growth and development. This naturally-occurring single-chainpituitary hormone consists of 191 amino acid residues and has amolecular weight of approximately 22 kDa. hGH exhibits a multitude ofbiological effects, including linear growth (somatogenesis), lactation,activation of macrophages, and insulin-like and diabetogenic effects,among others (Chawla, R., et al, Ann. Rev. Med. 34:519-547 (1983);Isaksson, O., et al., Ann. Rev. Physiol., 47:483-499 (1985); Hughes, J.and Friesen, H., Ann. Rev. Physiol., 47:469-482 (1985)). The structureof hGH is well known (Goeddel, D., et al., Nature 281:544-548 (1979)),and the three-dimensional structure of hGH has been solved by x-raycrystallography (de Vos, A., et al., Science 255:306-312 (1992)). Theprotein has a compact globular structure, comprising four amphipathicalpha helical bundles, termed A-D beginning from the N-terminus, whichare joined by loops. hGH also contains four cysteine residues, whichparticipate in two intramolecular disulfide bonds: C53 is paired withC165 and C182 is paired with C189. The hormone is not glycosylated andhas been expressed in a secreted form in E. coli (Chang, C., et al.,Gene 55:189-196 (1987)).

A number of naturally occurring mutants of hGH have been identified.These include hGH-V (Seeburg, DNA 1: 239 (1982); U.S. Pat. Nos.4,446,235, 4,670,393, and 4,665,180, which are incorporated by referenceherein) and a 20-kDa hGH containing a deletion of residues 32-46 of hGH(Kostyo et al., Biochem. Biophys. Acta 925: 314 (1987); Lewis, U., etal., J. Biol. Chem., 253:2679-2687 (1978)). In addition, numerous hGHvariants, arising from post-transcriptional, post-translational,secretory, metabolic processing, and other physiological processes, havebeen reported (Baumann, G., Endocrine Reviews 12: 424 (1991)). Thebiological effects of hGH derive from its interaction with specificcellular receptors. The hormone is a member of a family of homologousproteins that include placental lactogens and prolactins. hGH is unusualamong the family members, however, in that it exhibits broad speciesspecificity and binds to either the cloned somatogenic (Leung, D., etal., Nature 330:537-543 (1987)) or prolactin (Boutin, J., et al., Cell53:69-77 (1988)) receptor. Based on structural and biochemical studies,functional maps for the lactogenic and somatogenic binding domains havebeen proposed (Cunningham, B. and Wells, J., Proc. Natl. Acad. Sci. 88:3407 (1991)). The hGH receptor is a member of thehematopoietic/cytokine/growth factor receptor family, which includesseveral other growth factor receptors, such as the interleukin (IL)-3,-4 and -6 receptors, the granulocyte macrophage colony-stimulatingfactor (GM-CSF) receptor, the erythropoietin (EPO) receptor, as well asthe G-CSF receptor. See, Bazan, Proc. Natl. Acad. Sci. USA 87: 6934-6938(1990). Members of the cytokine receptor family contain four conservedcysteine residues and a tryptophan-serine-X-tryptophan-serine motifpositioned just outside the transmembrane region. The conservedsequences are thought to be involved in protein-protein interactions.See, e.g., Chiba et al., Biochim. Biophys. Res. Comm. 184: 485-490(1992). The interaction between hGH and extracellular domain of itsreceptor (hGHbp) is among the most well understood hormone-receptorinteractions. High-resolution X-ray crystallographic data (Cunningham,B., et al., Science, 254:821-825 (1991)) has shown that hGH has tworeceptor binding sites and binds two receptor molecules sequentiallyusing distinct sites on the molecule. The two receptor binding sites arereferred to as Site I and Site II. Site I includes the carboxy terminalend of helix D and parts of helix A and the A-B loop, whereas Site IIencompasses the amino terminal region of helix A and a portion of helixC. Binding of GH to its receptor occurs sequentially, with Site Ibinding first. Site II then engages a second GH receptor, resulting inreceptor dimerization and activation of the intracellular signalingpathways that lead to cellular responses to the hormone. An hGH muteinin which a G120R substitution has been introduced into site II is ableto bind a single hGH receptor, but is unable to dimerize two receptors.The mutein acts as an hGH antagonist in vitro, presumably by occupyingreceptor sites without activating intracellular signaling pathways (Fuh,G., et al., Science 256:1677-1680 (1992)).

Recombinant hGH is used as a therapeutic and has been approved for thetreatment of a number of indications, hGH deficiency leads to dwarfism,for example, which has been successfully treated for more than a decadeby exogenous administration of the hormone. Forms of hGH deficiency(GHD) include pediatric GHD, adult GHD of childhood onset, and adult GHDof adult onset. In addition to hGH deficiency, hGH has also beenapproved for the treatment of renal failure (in children), Turner'sSyndrome, and cachexia in AIDS patients. Recently, the Food and DrugAdministration (FDA) has approved hGH for the treatment ofnon-GH-dependent short stature. hGH is also currently underinvestigation for the treatment of aging, frailty in the elderly, shortbowel syndrome, and congestive heart failure. Target populations for hGHtreatment include children with idiopathic short stature (ISS) andadults with GHD-like symptoms. Recombinant hGH is currently sold as adaily injectable product, with five major products currently on themarket: HUMATROPE® (somatropin, Eli Lilly & Co.), NUTROPIN® (somatropin,Genentech), NORDITROPIN® (somatropin, Novo-Nordisk), GENOTROPIN®(somatropin, Pfizer) and SAIZEN®/SEROSTIM® (somatropin, Serono). Asignificant challenge to using growth hormone as a therapeutic, however,is that the protein has a short in vivo half-life and, therefore, itmust be administered by daily subcutaneous injection for maximumeffectiveness (MacGillivray, et al., J. Clin. Endocrinol. Metab. 81:1806-1809 (1996)). Considerable effort is focused on means to improvethe administration of hGH agonists and antagonists, by lowering the costof production, making administration easier for the patient, improvingefficacy and safety profile, and creating other properties that wouldprovide a competitive advantage. For example, Genentech and Alkermesformerly marketed NUTROPTN® Depot, a depot formulation of hGH, forpediatric growth hormone deficiency. While the depot permits lessfrequent administration (once every 2-3 weeks rather than once daily),it is also associated with undesirable side effects, such as decreasedbioavailability and pain at the injection site and was withdrawn fromthe market in 2004. Another pegvisomant product (SOMAVERT® Pfizer), hasalso recently been approved by the FDA. SOMAVERT® is agenetically-engineered analogue of hGH that functions as a highlyselective growth hormone receptor antagonist indicated for the treatmentof acromegaly (van der Lely, et al., The Lancet 358: 1754-1759 (2001).Although several of the amino acid side chain residues in SOMAVERT® arederivatized with polyethylene glycol (PEG) polymers, the product isstill administered once-daily, indicating that the pharmaceuticalproperties are not optimal. In addition to PEGylation and depotformulations, other administration routes, including inhaled and oraldosage forms of hGH, are under early-stage pre-clinical and clinicaldevelopment and none have yet received approval from the FDA.Accordingly, there is a need for a polypeptide that exhibits growthhormone activity but that also provides a longer serum half-life and,therefore, more optimal therapeutic levels of hGH and an increasedtherapeutic half-life.

Recently, an entirely new technology in the protein sciences has beenreported, which promises to overcome many of the limitations associatedwith site-specific modifications of proteins such as hGH. Specifically,new components have been added to the protein biosynthetic machinery ofthe prokaryote Escherichia coli (E. coli) (e.g., L. Wang, et al.,(2001), Science 292:498-500) and the eukaryote Saccharomyces cerevisiae(S. cerevisiae) (e.g., J. Chin et al., Science 301:964-7 (2003)), whichhas enabled the incorporation of non-genetically encoded amino acids toproteins in vivo. Constructs provided to host cells contain apolynucleotide encoding the hGH polypeptide comprising a selector codonand an orthogonal tRNA synthetase and/or an orthogonal tRNA forsubstituting a non-naturally encoded amino acid into the hGHpolypeptide. A number of new amino acids with novel chemical, physicalor biological properties, including photoaffinity labels andphotoisomerizable amino acids, photocrosslinking amino acids (see, e.g.,Chin, J. W., et al. (2002) Proc. Natl. Acad. Sci. U.S.A. 99:11020-11024;and, Chin, J. W., et al., (2002) J. Am. Chem. Soc. 124:9026-9027), ketoamino acids, heavy atom containing amino acids, and glycosylated aminoacids have been incorporated efficiently and with high fidelity intoproteins in E. coli and in yeast in response to the amber codon, TAG,using this methodology. See, e.g., J. W. Chin et al., (2002), Journal ofthe American Chemical Society 124:9026-9027; J. W. Chin, & P. G.Schultz, (2002), ChemBioChem 3(11):1135-1137; J. W. Chin, et al.,(2002), PNAS United States of America 99:11020-11024; and, L. Wang, & P.G. Schultz, (2002), Chem. Comm., 1:1-11. All references are incorporatedby reference herein in their entirety. These studies have demonstratedthat it is possible to selectively and routinely introduce chemicalfunctional groups that are chemically inert to all of the functionalgroups found in the 20 common, genetically-encoded amino acids and thatmay be used to react efficiently and selectively to form stable covalentlinkages. The ability to incorporate non-genetically encoded amino acidsinto proteins permits the introduction of chemical functional groupsthat could provide valuable alternatives to the naturally-occurringfunctional groups, such as the epsilon —NH₂ of lysine, the sulfhydryl—SH of cysteine, the imino group of histidine, etc. Certain chemicalfunctional groups are known to be inert to the functional groups foundin the 20 common, genetically-encoded amino acids but react cleanly andefficiently to form stable linkages.

Covalent attachment of the hydrophilic polymer poly(ethylene glycol),abbreviated PEG, is a method of increasing water solubility,bioavailability, increasing serum half-life, increasing therapeutichalf-life, modulating immunogenicity, modulating biological activity, orextending the circulation time of many biologically active molecules,including proteins, peptides, and particularly hydrophobic molecules.PEG has been used extensively in pharmaceuticals, on artificialimplants, and in other applications where biocompatibility, lack oftoxicity, and lack of immunogenicity are of importance. In order tomaximize the desired properties of PEG, the total molecular weight andhydration state of the PEG polymer or polymers attached to thebiologically active molecule must be sufficiently high to impart theadvantageous characteristics typically associated with PEG polymerattachment, such as increased water solubility and circulating halflife, while not adversely impacting the bioactivity of the parentmolecule. Any molecular mass for a PEG can be used as practicallydesired, including but not limited to, from about 100 Daltons (Da) to100,000 Da or more as desired (including but not limited to, sometimes0.1-50 kDa or 10-40 kDa). Branched chain PEGs, including but not limitedto, PEG molecules with each chain having a MW ranging from 1-100 kDa(including but not limited to, 1-50 kDa or 5-20 kDa) can also be used.

PEG derivatives are frequently linked to biologically active moleculesthrough reactive chemical functionalities, such as lysine, cysteine andhistidine residues, the N-terminus and carbohydrate moieties. Proteinsand other molecules often have a limited number of reactive sitesavailable for polymer attachment. Often, the sites most suitable formodification via polymer attachment play a significant role in receptorbinding, and are necessary for retention of the biological activity ofthe molecule. As a result, indiscriminate attachment of polymer chainsto such reactive sites on a biologically active molecule often leads toa significant reduction or even total loss of biological activity of thepolymer-modified molecule. R. Clark et al., (1996), J. Biol. Chem.,271:21969-21977. To form conjugates having sufficient polymer molecularweight for imparting the desired advantages to a target molecule, priorart approaches have typically involved random attachment of numerouspolymer arms to the molecule, thereby increasing the risk of a reductionor even total loss in bioactivity of the parent molecule.

Reactive sites that form the loci for attachment of PEG derivatives toproteins are dictated by the protein's structure. Proteins, includingenzymes, are composed of various sequences of alpha-amino acids, whichhave the general structure H₂N—CHR—COOH. The alpha amino moiety (H₂N—)of one amino acid joins to the carboxyl moiety (—COOH) of an adjacentamino acid to form amide linkages, which can be represented as—(NH—CHR—CO)_(n)—, where the subscript “n” can equal hundreds orthousands. The fragment represented by R can contain reactive sites forprotein biological activity and for attachment of PEG derivatives.

For example, in the case of the amino acid lysine, there exists an —NH₂moiety in the epsilon position as well as in the alpha position. Theepsilon —NH₂ is free for reaction under conditions of basic pH. Much ofthe art in the field of protein derivatization with PEG has beendirected to developing PEG derivatives for attachment to the epsilon—NH₂ moiety of lysine residues present in proteins. “Polyethylene Glycoland Derivatives for Advanced PEGylation”, Nektar Molecular EngineeringCatalog, 2003, pp. 1-17. These PEG derivatives all have the commonlimitation, however, that they cannot be installed selectively among theoften numerous lysine residues present on the surfaces of proteins. Thiscan be a significant limitation in instances where a lysine residue isimportant to protein activity, existing in an enzyme active site forexample, or in cases where a lysine residue plays a role in mediatingthe interaction of the protein with other biological molecules, as inthe case of receptor binding sites.

A second and equally important complication of existing methods forprotein PEGylation is that the PEG derivatives can undergo undesiredside reactions with residues other than those desired. Histidinecontains a reactive imino moiety, represented structurally as—N(H)—, butmany chemically reactive species that react with epsilon —NH₂ can alsoreact with N(H)—. Similarly, the side chain of the amino acid cysteinebears a free sulfhydryl group, represented structurally as —SH. In someinstances, the PEG derivatives directed at the epsilon —NH₂ group oflysine also react with cysteine, histidine or other residues. This cancreate complex, heterogeneous mixtures of PEG-derivatized bioactivemolecules and risks destroying the activity of the bioactive moleculebeing targeted. It would be desirable to develop PEG derivatives thatpermit a chemical functional group to be introduced at a single sitewithin the protein that would then enable the selective coupling of oneor more PEG polymers to the bioactive molecule at specific sites on theprotein surface that are both well-defined and predictable.

In addition to lysine residues, considerable effort in the art has beendirected toward the development of activated PEG reagents that targetother amino acid side chains, including cysteine, histidine and theN-terminus. See, e.g., U.S. Pat. No. 6,610,281 which is incorporated byreference herein, and “Polyethylene Glycol and Derivatives for AdvancedPEGylation”, Nektar Molecular Engineering Catalog, 2003, pp. 1-17. Acysteine residue can be introduced site-selectively into the structureof proteins using site-directed mutagenesis and other techniques knownin the art, and the resulting free sulfhydryl moiety can be reacted withPEG derivatives that bear thiol-reactive functional groups. Thisapproach is complicated, however, in that the introduction of a freesulfhydryl group can complicate the expression, folding and stability ofthe resulting protein. Thus, it would be desirable to have a means tointroduce a chemical functional group into bioactive molecules thatenables the selective coupling of one or more PEG polymers to theprotein while simultaneously being compatible with (i.e., not engagingin undesired side reactions with) sulfhydryls and other chemicalfunctional groups typically found in proteins.

As can be seen from a sampling of the art, many of these derivativesthat have been developed for attachment to the side chains of proteins,in particular, the —NH₂ moiety on the lysine amino acid side chain andthe —SH moiety on the cysteine side chain, have proven problematic intheir synthesis and use. Some form unstable linkages with the proteinthat are subject to hydrolysis and therefore decompose, degrade, or areotherwise unstable in aqueous environments, such as in the bloodstream.Some form more stable linkages, but are subject to hydrolysis before thelinkage is formed, which means that the reactive group on the PEGderivative may be inactivated before the protein can be attached. Someare somewhat toxic and are therefore less suitable for use in vivo. Someare too slow to react to be practically useful. Some result in a loss ofprotein activity by attaching to sites responsible for the protein'sactivity. Some are not specific in the sites to which they will attach,which can also result in a loss of desirable activity and in a lack ofreproducibility of results. In order to overcome the challengesassociated with modifying proteins with poly(ethylene glycol) moieties,PEG derivatives have been developed that are more stable (e.g., U.S.Pat. No. 6,602,498, which is incorporated by reference herein) or thatreact selectively with thiol moieties on molecules and surfaces (e.g.,U.S. Pat. No. 6,610,281, which is incorporated by reference herein).There is clearly a need in the art for PEG derivatives that arechemically inert in physiological environments until called upon toreact selectively to form stable chemical bonds.

Therefore, there currently exists an unmet need to provide hGHpolypeptide in a substantially pure form suitable for use in humantherapeutic applications. In addition, methods for the production ofpharmaceutical grade hGH polypeptide are needed that are amenable tolarge-scale production that are highly efficient and cost productive.

BRIEF SUMMARY OF THE INVENTION

The present invention relates generally to the production andpurification of hGH polypeptide from recombinant host cells or culturemedia. More particularly, the invention relates to the production andpurification of substantially purified hGH polypeptide from arecombinant host, including, but not limited to, a prokaryotic host, abacterial host or an E. coli host.

In one embodiment, the present invention provides methods for isolatingsubstantially purified hGH polypeptide comprising the steps of: (a)anion exchange chromatography; and (b) hydrophobic interactionchromatography (HIC). Purification of PEGylated hGH polypeptide includesthe following additional steps: (c) reacting hGH polypeptide with PEG toform hGH-PEG conjugates; and (d) isolating said hGH-PEG conjugates by ananion exchange chromatography.

In another embodiment, the present invention provides methods forisolating substantially purified hGH polypeptide comprising the stepsof: (a) anion exchange chromatography; (b) hydroxyapatitechromatography; and (c) hydrophobic interaction chromatography (HIC).Purification of PEGylated hGH polypeptide includes the followingadditional steps: (d) reacting hGH polypeptide with PEG to form hGH-PEGconjugates; and (e) isolating said hGH-PEG conjugates by an anionexchange chromatography.

In one embodiment, the recombinant host is selected from the groupconsisting of a prokaryotic cell and a eukaryotic cell. In oneembodiment, the recombinant host may comprise any host cell thatproduces an insoluble sub-cellular component, such as inclusion bodies,comprising hGH polypeptide including, for example, yeast cells,mammalian cells, insect cells and bacterial cells, including, forexample, E. coli.

The hydrophobic interaction chromatography materials suitable for use inthe methods of the present invention may include, but are not limitedto, alkyl- or aryl-substituted matrices, such as butyl-, hexyl-, octyl-or phenyl-substituted matrices including agarose, cross-linked agarose,sepharose, cellulose, silica, dextran, polystyrene, poly(methacrylate)matrices, and mixed mode resins, including but not limited to, apolyethyleneamine resin or a butyl- or phenyl-substitutedpoly(methacrylate) matrix. In a specific embodiment, the hydrophobicinteraction chromatography material may comprise phenyl sepharose resin.

In one embodiment, the substantially purified hGH polypeptide isolatedby the methods described herein may include, but is not limited to,mature hGH, mature hGH variants, hGH polypeptides, hGH polypeptidevariants, and hGH conjugated to poly(ethylene glycol).

In yet another embodiment, the substantially purified hGH polypeptideisolated by the methods of the present invention may exhibit at leastone biological activity of mature hGH.

In yet another embodiment, the substantially purified hGH polypeptideisolated by the methods of the present invention may be mammalian. In aspecific embodiment, the substantially purified hGH polypeptide isolatedby the methods of the present invention may be human.

In another embodiment of the present invention, the hGH polypeptideobtained from the HIC step is covalently linked to a water solublepolymer. In some embodiments, the water soluble polymer is poly(ethyleneglycol). In another embodiment, the hGH polypeptide comprises one ormore non-naturally encoded amino acids.

Expression of hGH polypeptides comprising a non-naturally encoded aminoacid and purification of PEGylated forms thereof provide hGH moleculesaltered in a site-specific manner for therapeutic use. PEGylation of hGHpolypeptides at naturally encoded amino acids may result in thePEGylation of hGH polypeptide at undesired sites and/or PEGylation ofundesired polypeptides that may be contaminants. Methods utilizingnon-naturally encoded amino acids for site-specific PEGylation of hGHpolypeptide renders such purification steps unnecessary.

In another embodiment, conjugation of the hGH polypeptide comprising oneor more non-naturally occurring amino acids to another molecule,including but not limited to PEG, provides substantially purified hGHpolypeptide due to the unique chemical reaction utilized for conjugationto the non-natural amino acid. Conjugation of hGH polypeptide comprisingone or more non-naturally encoded amino acids to another molecule, suchas PEG, may be performed with other purification techniques performedprior to or following the conjugation step to provide substantially purehGH polypeptide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows feed flow rates for an 8 liter fermentation.

FIG. 2 shows a fermentation process on a 5 liter scale.

FIG. 3, Panels A and B show SDS-PAGE analysis of hGH polypeptideprepared by periplasmic release and homogenization.

FIG. 4 shows a process flow for a 5 liter fermentation.

FIG. 5 shows a chemical structure of a linear, 30 kDa PEG.

DEFINITIONS

It is to be understood that this invention is not limited to theparticular methodology, protocols, cell lines, constructs, and reagentsdescribed herein and as such may vary. It is also to be understood thatthe terminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention, which will be limited only by the appended claims.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural reference unless the context clearly indicatesotherwise. Thus, for example, reference to a “hGH” is a reference to oneor more such proteins and includes equivalents thereof known to those ofordinary skill in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs. Although any methods, devices,and materials similar or equivalent to those described herein can beused in the practice or testing of the invention, the preferred methods,devices and materials are now described.

All publications and patents mentioned herein are incorporated herein byreference for the purpose of describing and disclosing, for example, theconstructs and methodologies that are described in the publications,which might be used in connection with the presently describedinvention. The publications discussed herein are provided solely fortheir disclosure prior to the filing date of the present application.Nothing herein is to be construed as an admission that the inventors arenot entitled to antedate such disclosure by virtue of prior invention orfor any other reason.

U.S. patent application Ser. No. 11/046,432 is incorporated by referenceherein in its entirety. U.S. patent application Ser. No. 11/046,432describes the naturally-occurring amino acid sequences of hGH, siteselection for incorporation of non-naturally encoded amino acids, andmethods, compositions, techniques and strategies for making, purifying,characterizing, and using non-naturally encoded amino acids,non-naturally encoded amino acid hGH polypeptides, and modifiednon-naturally encoded amino acid hGH polypeptides.

The term “protein” as used herein, includes a polymer or complex ofvarious polymers of amino acids and does not connote a specific lengthof a polymer of amino acids. Thus, for example, the terms peptide,oligopeptide, and polypeptide are included within the definition ofprotein, whether produced using recombinant techniques, chemical orenzymatic synthesis, or naturally occurring. The term also includespeptides, oligopeptides, and polypeptides that have been modified orderivatized, such as by glycosylation, acetylation, phosphorylation, andthe like. The term “protein” specifically includes variants, as definedherein. The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues.That is, a description directed to a polypeptide applies equally to adescription of a peptide and a description of a protein, and vice versa.The terms apply to naturally occurring amino acid polymers as well asamino acid polymers in which one or more amino acid residues is anon-naturally encoded amino acid. As used herein, the terms encompassamino acid chains of any length, including full length proteins, whereinthe amino acid residues are linked by covalent peptide bonds.

As used herein, “growth hormone” or “GH” shall include thosepolypeptides and proteins that have at least one biological activity ofa human growth hormone, as well as GH analogs, GH isoforms, GH mimetics,GH fragments, hybrid GH proteins, fusion proteins, oligomers andmultimers, homologues, glycosylation pattern variants, variants, splicevariants, and muteins, thereof, regardless of the biological activity ofsame, and further regardless of the method of synthesis or manufacturethereof including, but not limited to, recombinant (whether producedfrom cDNA, genomic DNA, synthetic DNA or other form of nucleic acid), invitro, in vivo, by microinjection of nucleic acid molecules, synthetic,transgenic, and gene activated methods. The term “hGH polypeptide” or“hGH” encompasses hGH polypeptides comprising one or more amino acidsubstitutions, additions or deletions. Exemplary substitutions in a widevariety of amino acid positions in naturally-occurring hGH includingsubstitutions that increase agonist activity, increase proteaseresistance, convert the polypeptide into an antagonist, modulateimmunogenicity, modulate receptor binding, etc. are encompassed by theterm “hGH polypeptide.”

For the complete full-length naturally-occurring GH amino acid sequenceas well as the mature naturally-occurring GH amino acid sequence andnaturally occurring mutant, see SEQ ID NO: 1, SEQ ID NO: 2 and SEQ IDNO: 3, respectively, herein. In some embodiments, hGH polypeptides ofthe invention are substantially identical to SEQ ID NO: 1, or SEQ ID NO:2, or SEQ ID NO: 3 or any other sequence of a growth hormonepolypeptide. A number of naturally occurring mutants of hGH have beenidentified. These include hGH-V (Seeburg, DNA 1: 239 (1982); U.S. Pat.Nos. 4,446,235, 4,670,393, and 4,665,180, which are incorporated byreference herein) and a 20-kDa hGH containing a deletion of residues32-46 of hGH (Kostyo et al., Biochem. Biophys. Acta 925: 314 (1987);Lewis, U., et al., J. Biol. Chem., 253:2679-2687 (1978)). Placentalgrowth hormone is described in Igout, A., et al., Nucleic Acids Res.17(10):3998 (1989)). In addition, numerous hGH variants, arising frompost-transcriptional, post-translational, secretory, metabolicprocessing, and other physiological processes, have been reportedincluding proteolytically cleaved or 2 chain variants (Baumann, G.,Endocrine Reviews 12: 424 (1991)). Nucleic acid molecules encoding hGHmutants and mutant hGH polypeptides are well known and include, but arenot limited to, those disclosed in U.S. Pat. Nos. 5,534,617; 5,580,723;5,688,666; 5,750,373; 5,834,250; 5,834,598; 5,849,535; 5,854,026;5,962,411; 5,955,346; 6,013,478; 6,022,711; 6,136,563; 6,143,523;6,428,954; 6,451,561; 6,780,613 and U.S. Patent Application Publication2003/0153003; which are incorporated by reference herein. The term“^(a)hGH” may also be used to refer to recombinant human growth hormonewith site-directed substitution of a non-naturally encoded amino acid.

All references to amino acid positions in hGH described herein are basedon the position in SEQ ID NO: 2, unless otherwise specified (i.e., whenit is stated that the comparison is based on SEQ ID NO: 1, 3, or otherhGH sequence). Those of skill in the art will appreciate that amino acidpositions corresponding to positions in SEQ ID NO: 1, 2, 3, or any otherGH sequence can be readily identified in any other hGH molecule such ashGH fusions, variants, fragments, etc. For example, sequence alignmentprograms such as BLAST can be used to align and identify a particularposition in a protein that corresponds with a position in SEQ ID NO: 1,2, 3, or other GH sequence. Substitutions, deletions or additions ofamino acids described herein in reference to SEQ ID NO: 1, 2, 3, orother GH sequence are intended to also refer to substitutions, deletionsor additions in corresponding positions in hGH fusions, variants,fragments, etc. described herein or known in the art and are expresslyencompassed by the present invention.

Commercial preparations of hGH are sold under the names: HUMATROPE®(somatropin, Eli Lilly & Co.), NUTROPIN® (somatropin, Genentech),NORDITROPIN® (somatropin, Novo-Nordisk), GENOTROPIN® (somatropin,Pfizer) and SAIZEN®/SEROSTIM® (somatropin, Serono).

The term “hGH polypeptide” also includes the pharmaceutically acceptablesalts and prodrugs, and prodrugs of the salts, polymorphs, hydrates,solvates, biologically-active fragments, biologically active variantsand stereoisomers of the naturally-occurring hGH as well as agonist,mimetic, and antagonist variants of the naturally-occurring hGH andpolypeptide fusions thereof. Fusions comprising additional amino acidsat the amino terminus, carboxyl terminus, or both, are encompassed bythe term “hGH polypeptide.” Exemplary fusions include, but are notlimited to, e.g., methionyl growth hormone in which a methionine islinked to the N-terminus of hGH resulting from the recombinantexpression, fusions for the purpose of purification (including, but notlimited to, to poly-histidine or affinity epitopes), fusions with serumalbumin binding peptides and fusions with serum proteins such as serumalbumin. U.S. Pat. No. 5,750,373, which is incorporated by referenceherein, describes a method for selecting novel proteins such as growthhormone and antibody fragment variants having altered binding propertiesfor their respective receptor molecules. The method comprises fusing agene encoding a protein of interest to the carboxy terminal domain ofthe gene III coat protein of the filamentous phage M13.

Various references disclose modification of polypeptides by polymerconjugation or glycosylation. The term “hGH polypeptide” includespolypeptides conjugated to a polymer such as PEG and may be comprised ofone or more additional derivitizations of cysteine, lysine, or otherresidues. In addition, the hGH polypeptide may comprise a linker orpolymer, wherein the amino acid to which the linker or polymer isconjugated may be a non-natural amino acid according to the presentinvention, or may be conjugated to a naturally encoded amino acidutilizing techniques known in the art such as coupling to lysine orcysteine.

The present invention provides conjugates of hGH polypeptide having awide variety of functional groups, substituents or moieties, with othersubstances including but not limited to a label; a dye; a polymer; awater-soluble polymer; a derivative of polyethylene glycol; aphotocrosslinker; a radionuclide; a cytotoxic compound; a drug; anaffinity label; a photoaffinity label; a reactive compound; a resin; asecond protein or polypeptide or polypeptide analog; an antibody orantibody fragment; a metal chelator; a cofactor; a fatty acid; acarbohydrate; a polynucleotide; a DNA; a RNA; an antisensepolynucleotide; a saccharide; a water-soluble dendrimer; a cyclodextrin;an inhibitory ribonucleic acid; a biomaterial; a nanoparticle; a spinlabel; a fluorophore, a metal-containing moiety; a radioactive moiety; anovel functional group; a group that covalently or noncovalentlyinteracts with other molecules; a photocaged moiety; a photoisomerizablemoiety; biotin; a derivative of biotin; a biotin analogue; a moietyincorporating a heavy atom; a chemically cleavable group; aphotocleavable group; an elongated side chain; a carbon-linked sugar; aredox-active agent; an amino thioacid; a toxic moiety; an isotopicallylabeled moiety; a biophysical probe; a phosphorescent group; achemiluminescent group; an electron dense group; a magnetic group; anintercalating group; a chromophore; an energy transfer agent; abiologically active agent; a detectable label; a small molecule; aquantum dot, a nanotransmitter, a radionucleotide, a radiotransmitter, aneutron-capture agent, or any combination of the above, or any otherdesirable compound or substance).

Polymer conjugation of hGH polypeptides has been reported. See, e.g.U.S. Pat. Nos. 5,849,535, 6,136,563 and 6,608,183, which areincorporated by reference herein. U.S. Pat. No. 4,904,584 disclosesPEGylated lysine depleted polypeptides, wherein at least one lysineresidue has been deleted or replaced with any other amino acid residue.WO 99/67291 discloses a process for conjugating a protein with PEG,wherein at least one amino acid residue on the protein is deleted andthe protein is contacted with PEG under conditions sufficient to achieveconjugation to the protein. WO 99/03887 discloses PEGylated variants ofpolypeptides belonging to the growth hormone superfamily, wherein acysteine residue has been substituted with a non-essential amino acidresidue located in a specified region of the polypeptide. WO 00/26354discloses a method of producing a glycosylated polypeptide variant withreduced allergenicity, which as compared to a corresponding parentpolypeptide comprises at least one additional glycosylation site. U.S.Pat. No. 5,218,092 discloses modification of granulocyte colonystimulating factor (G-CSF) and other polypeptides so as to introduce atleast one additional carbohydrate chain as compared to the nativepolypeptide.

The term “hGH polypeptide” encompasses hGH polypeptides comprising oneor more amino acid substitutions, additions or deletions. hGHpolypeptides of the present invention may be comprised of modificationswith one or more natural amino acids in conjunction with one or morenon-natural amino acid modification. Exemplary substitutions in a widevariety of amino acid positions in naturally-occurring hGH polypeptideshave been described, including but not limited to substitutions thatmodulate one or more of the biological activities of the hGHpolypeptide, such as but not limited to, increase agonist activity,increase solubility of the polypeptide, convert the polypeptide into anantagonist, decrease protease susceptibility, etc. and are encompassedby the term “hGH polypeptide.” In some embodiments, the hGH polypeptidesfurther comprise an addition, substitution or deletion that modulatesbiological activity of the hGH polypeptide. For example, the additions,substitutions or deletions may modulate affinity for the hGH polypeptidereceptor, modulate (including but not limited to, increases ordecreases) receptor dimerization, stabilize receptor dimers, modulatecirculating half-life, modulate therapeutic half-life, modulatestability of the polypeptide, modulate cleavage by proteases, modulatedose, modulate release or bio-availability, facilitate purification, orimprove or alter a particular route of administration.

Similarly, hGH polypeptides may comprise protease cleavage sequences,reactive groups, antibody-binding domains (including but not limited to,FLAG or poly-His) or other affinity based sequences (including but notlimited to, FLAG, poly-His, GST, etc.) or linked molecules (includingbut not limited to, biotin) that improve detection (including but notlimited to, GFP), purification or other traits of the polypeptide. hGHpolypeptides may comprise secretion signal sequences. Examples ofsecretion signal sequences include, but are not limited to, aprokaryotic secretion signal sequence, an eukaryotic secretion signalsequence, an eukaryotic secretion signal sequence 5′-optimized forbacterial expression, a novel secretion signal sequence, pectate lyasesecretion signal sequence, Omp A secretion signal sequence, and a phagesecretion signal sequence. Examples of secretion signal sequences,include, but are not limited to, STII (prokaryotic), Fd GIII and M13(phage), Bgl2 (yeast), and the signal sequence bla derived from atransposon.

The term “hGH polypeptide” also encompasses homodimers, heterodimers,homomultimers, and heteromultimers that are linked, including but notlimited to those linked directly via non-naturally encoded amino acidside chains, either to the same or different non-naturally encoded aminoacid side chains, to naturally-encoded amino acid side chains, orindirectly via a linker. hGH dimers linked directly via Cys-Cysdisulfide linkages are described in Lewis, U. J., et al., J. Biol. Chem.252:3697-3702 (1977); Brostedt, P. and Roos, P., Prep. Biochem.19:217-229 (1989)). Exemplary linkers including but are not limited to,water soluble polymers such as poly(ethylene glycol) or polydextran orpolypeptides of various lengths.

The term “hGH polypeptide” also includes glycosylated hGH, such as butnot limited to, polypeptides glycosylated at any amino acid position,N-linked or O-linked glycosylated forms of the polypeptide. Variantscontaining single nucleotide changes are also considered as biologicallyactive variants of hGH polypeptide. In addition, splice variants arealso included. The term “hGH polypeptide” also includes hGH polypeptideheterodimers, homodimers, heteromultimers, or homomultimers of any oneor more hGH polypeptides or any other polypeptide, protein,carbohydrate, polymer, small molecule, linker, ligand, or otherbiologically active molecule of any type, linked by chemical means orexpressed as a fusion protein, as well as polypeptide analoguescontaining, for example, specific deletions or other modifications yetmaintain biological activity.

Those of skill in the art will appreciate that amino acid positionscorresponding to positions in a particular hGH sequence can be readilyidentified in any other hGH molecule such as hGH fusions, variants,fragments, etc. For example, sequence alignment programs such as BLASTcan be used to align and identify a particular position in a proteinthat corresponds with a position in a particular GH sequence.

“Native hGH,” as used herein, is defined as hGH, including naturallyoccurring hGH, analogs, and variants thereof, which is properly foldedand contains only correct disulfide bonds. hGH also contains fourcysteine residues, which participate in two intramolecular disulfidebonds: C53 is paired with C165 and C182 is paired with C189 or thehomologs of those amino acid residues in analogs and variants of hGH.Native hGH is biologically active.

“Insoluble hGH” refers to precipitated or aggregated hGH that isproduced by recombinant host cells, or is otherwise recombinant hostcell associated, and may assume a biologically inactive conformationwith possible incorrect or unformed disulfide bonds. Insoluble hGH maybe contained in inclusion bodies or refractile bodies, i.e. may or maynot be visible under a phase contrast microscope. Insoluble hGH may beproduced by rendering soluble hGH insoluble by any method known to oneof ordinary skill in the art.

“Improperly folded hGH” refers to hGH which is in a biologically lessactive conformation with incorrect or unformed disulfide bonds.Improperly folded hGH may be, but need not be, insoluble.

The term “hGH variant,” as used herein, includes variants of mature hGHand hGH polypeptides. A “hGH variant” may be created by, and includes,for example, the deletion or addition of one or more amino acids at oneor more sites in the mature protein, deletion or addition of one or moreamino acids to the N-terminal and/or C-terminal end of the matureprotein, and/or substitution of one or more amino acids at one or moresites in the mature protein. For example, a hGH variant may be createdby adding or deleting at least 10 amino acids, at least 5 amino acids,at least 3 amino acids, or at least 1 amino acid. hGH variants may alsoinclude post-translational modifications including, but not limited to,glycosylation, acetylation, phosphorylation, and the like. The term “hGHvariant” specifically includes, but is not limited to, mutants, allelicvariants, homologs, and fusions of mature hGH sequences. An hGH variantalso includes, but is not limited to, peptide mimics or “peptoids.” SeeWO 91/04282.

The term “substantially purified” refers to hGH polypeptide that may besubstantially or essentially free of components that normally accompanyor interact with the protein as found in its naturally occurringenvironment, i.e. a native cell, or host cell in the case ofrecombinantly produced hGH polypeptide. hGH that may be substantiallyfree of cellular material includes preparations of protein having lessthan about 30%, less than about 25%, less than about 20%, less thanabout 15%, less than about 10%, less than about 5%, less than about 4%,less than about 3%, less than about 2%, or less than about 1% (by dryweight) of contaminating protein. When the hGH polypeptide or variantthereof is recombinantly produced by the host cells, the protein may bepresent at about 30%, about 25%, about 20%, about 15%, about 10%, about5%, about 4%, about 3%, about 2%, or about 1% or less of the dry weightof the cells. When the hGH polypeptide or variant thereof isrecombinantly produced by the host cells, the protein may be present inthe culture medium at about 5 g/L, about 4 g/L, about 3 g/L, about 2g/L, about 1 g/L, about 750 mg/L, about 500 mg/L, about 250 mg/L, about100 mg/L, about 50 mg/L, about 10 mg/L, or about 1 mg/L or less of thedry weight of the cells. Thus, “substantially purified” hGH polypeptideas produced by the methods of the present invention may have a puritylevel of at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, at least about 55%, at least about60%, at least about 65%, at least about 70%, specifically, a puritylevel of at least about 75%, 80%, 85%, and more specifically, a puritylevel of at least about 90%, a purity level of at least about 95%, apurity level of at least about 99% or greater, as determined byappropriate methods including, but not limited to, SDS/PAGE analysis,RP-HPLC, SEC, and capillary electrophoresis.

A “recombinant host cell” or “host cell” refers to a cell that includesan exogenous polynucleotide, regardless of the method used forinsertion, for example, direct uptake, transduction, f-mating, or othermethods known in the art to create recombinant host cells. The exogenouspolynucleotide may be maintained as a nonintegrated vector, for example,a plasmid, or alternatively, may be integrated into the host genome.

As used herein, the term “medium” or “media” includes any culturemedium, solution, solid, semi-solid, or rigid support that may supportor contain any host cell, including bacterial host cells, eukaryotichost cells, mammalian host cells, yeast host cells, insect host cells,plant host cells, CHO cells, prokaryotic host cells, E. coli, orPseudomonas host cells, and cell contents. Thus, the term may encompassmedium in which the host cell has been grown, e.g., medium into whichthe hGH polypeptide has been secreted, including medium either before orafter a proliferation step. The term also may encompass buffers orreagents that contain host cell lysates, such as in the case where hGHpolypeptides are produced intracellularly and the host cells are lysedor disrupted to release the hGH polypeptide.

“Reducing agent,” as used herein with respect to protein refolding, isdefined as any compound or material which maintains sulfhydryl groups inthe reduced state and reduces intra- or intermolecular disulfide bonds.Suitable reducing agents include, but are not limited to, dithiothreitol(DTT), 2-mercaptoethanol, dithioerythritol, cysteine, cysteamine(2-aminoethanethiol), and reduced glutathione. It is readily apparent tothose of ordinary skill in the art that a wide variety of reducingagents are suitable for use in the methods of the present invention.

“Oxidizing agent,” as used herein with respect to protein refolding, isdefined as any compound or material which is capable of removing anelectron from a compound being oxidized. Suitable oxidizing agentsinclude, but are not limited to, oxidized glutathione, cystine,cystamine, oxidized dithiothreitol, oxidized erythreitol, and oxygen. Itis readily apparent to those of ordinary skill in the art that a widevariety of oxidizing agents are suitable for use in the methods of thepresent invention.

“Denaturing agent” or “denaturant,” as used herein, is defined as anycompound or material which will cause a reversible unfolding of aprotein. The strength of a denaturing agent or denaturant will bedetermined both by the properties and the concentration of theparticular denaturing agent or denaturant. Suitable denaturing agents ordenaturants may be chaotropes, detergents, organic solvents, watermiscible solvents, phospholipids, or a combination of two or more suchagents. Suitable chaotropes include, but are not limited to, urea,guanidine, and sodium thiocyanate. Useful detergents may include, butare not limited to, strong detergents such as sodium dodecyl sulfate, orpolyoxyethylene ethers (e.g. Tween or Triton detergents), Sarkosyl, mildnon-ionic detergents (e.g., digitonin), mild cationic detergents such asN->2,3-(Dioleyoxy)-propyl-N,N,N-trimethylammonium, mild ionic detergents(e.g. sodium cholate or sodium deoxycholate) or zwitterionic detergentsincluding, but not limited to, sulfobetaines (Zwittergent),3-(3-chlolamidopropyl)dimethylammonio-1-propane sulfate (CHAPS), and3-(3-chlolamidopropyl)dimethylammonio-2-hydroxy-1-propane sulfonate(CHAPSO). Organic, water miscible solvents such as acetonitrile, loweralkanols (especially C₂-C₄ alkanols such as ethanol or isopropanol), orlower alkandiols (especially C₂-C₄ alkandiols such as ethylene-glycol)may be used as denaturants. Phospholipids useful in the presentinvention may be naturally occurring phospholipids such asphosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, andphosphatidylinositol or synthetic phospholipid derivatives or variantssuch as dihexanoylphosphatidylcholine or diheptanoylphosphatidylcholine.

“Refolding,” as used herein describes any process, reaction or methodwhich transforms disulfide bond containing polypeptides from animproperly folded or unfolded state to a native or properly foldedconformation with respect to disulfide bonds.

“Cofolding,” as used herein, refers specifically to refolding processes,reactions, or methods which employ at least two polypeptides whichinteract with each other and result in the transformation of unfolded orimproperly folded polypeptides to native, properly folded polypeptides.

A “non-naturally encoded amino acid” refers to an amino acid that is notone of the 20 common amino acids or pyrrolysine or selenocysteine. Otherterms that may be used synonymously with the term “non-naturally encodedamino acid” are “non-natural amino acid,” “unnatural amino acid,”“non-naturally-occurring amino acid,” and variously hyphenated andnon-hyphenated versions thereof. The term “non-naturally encoded aminoacid” also includes, but is not limited to, amino acids that occur bymodification (e.g. post-translational modifications) of a naturallyencoded amino acid (including but not limited to, the 20 common aminoacids or pyrrolysine and selenocysteine) but are not themselvesnaturally incorporated into a growing polypeptide chain by thetranslation complex. Examples of such non-naturally-occurring aminoacids include, but are not limited to, N-acetylglucosaminyl-L-serine,N-acetylglucosaminyl-L-threonine, and O-phosphotyrosine.

As used herein, the term “water soluble polymer” refers to any polymerthat is soluble in aqueous solvents. Linkage of water soluble polymersto hGH polypeptides can result in changes including, but not limited to,increased or modulated serum half-life, or increased or modulatedtherapeutic half-life relative to the unmodified form, modulatedimmunogenicity, modulated physical association characteristics such asaggregation and multimer formation, altered receptor binding, andaltered receptor dimerization or multimerization. The water solublepolymer may or may not have its own biological activity, and may beutilized as a linker for attaching hGH to other substances, includingbut not limited to one or more hGH polypeptides, or one or morebiologically active molecules. Suitable polymers include, but are notlimited to, polyethylene glycol, polyethylene glycol propionaldehyde,mono C1-C10 alkoxy or aryloxy derivatives thereof (described in U.S.Pat. No. 5,252,714 which is incorporated by reference herein),monomethoxy-polyethylene glycol, polyvinyl pyrrolidone, polyvinylalcohol, polyamino acids, divinylether maleic anhydride,N-(2-Hydroxypropyl)-methacrylamide, dextran, dextran derivativesincluding dextran sulfate, polypropylene glycol, polypropyleneoxide/ethylene oxide copolymer, polyoxyethylated polyol, heparin,heparin fragments, polysaccharides, oligosaccharides, glycans, celluloseand cellulose derivatives, including but not limited to methylcelluloseand carboxymethyl cellulose, starch and starch derivatives,polypeptides, polyalkylene glycol and derivatives thereof, copolymers ofpolyalkylene glycols and derivatives thereof, polyvinyl ethyl ethers,and alpha-beta-poly[(2-hydroxyethyl)-DL-aspartamide, and the like, ormixtures thereof. Examples of such water soluble polymers include, butare not limited to, polyethylene glycol and serum albumin.

An “amino terminus modification group” refers to any molecule that canbe attached to the amino terminus of a polypeptide. Similarly, a“carboxy terminus modification group” refers to any molecule that can beattached to the carboxy terminus of a polypeptide. Terminus modificationgroups include, but are not limited to, various water soluble polymers,peptides or proteins such as serum albumin, or other moieties thatincrease serum half-life of peptides.

As used herein, the term “polyalkylene glycol” or “poly(alkene glycol)”refers to polyethylene glycol (poly(ethylene glycol)), polypropyleneglycol, polybutylene glycol, and derivatives thereof. The term“polyalkylene glycol” encompasses both linear and branched polymers andaverage molecular weights of between 0.1 kDa and 100 kDa. Otherexemplary embodiments are listed, for example, in commercial suppliercatalogs, such as Shearwater Corporation's catalog “Polyethylene Glycoland Derivatives for Biomedical Applications” (2001).

The terms “functional group”, “active moiety”, “activating group”,“leaving group”, “reactive site”, “chemically reactive group” and“chemically reactive moiety” are used in the art and herein to refer todistinct, definable portions or units of a molecule. The terms aresomewhat synonymous in the chemical arts and are used herein to indicatethe portions of molecules that perform some function or activity and arereactive with other molecules.

The term “linkage” or “linker” is used herein to refer to groups orbonds that normally are formed as the result of a chemical reaction andtypically are covalent linkages. Hydrolytically stable linkages meansthat the linkages are substantially stable in water and do not reactwith water at useful pH values, including but not limited to, underphysiological conditions for an extended period of time, perhaps evenindefinitely. Hydrolytically unstable or degradable linkages mean thatthe linkages are degradable in water or in aqueous solutions, includingfor example, blood. Enzymatically unstable or degradable linkages meanthat the linkage can be degraded by one or more enzymes. As understoodin the art, PEG and related polymers may include degradable linkages inthe polymer backbone or in the linker group between the polymer backboneand one or more of the terminal functional groups of the polymermolecule. For example, ester linkages formed by the reaction of PEGcarboxylic acids or activated PEG carboxylic acids with alcohol groupson a biologically active agent generally hydrolyze under physiologicalconditions to release the agent. Other hydrolytically degradablelinkages include, but are not limited to, carbonate linkages; iminelinkages resulted from reaction of an amine and an aldehyde; phosphateester linkages formed by reacting an alcohol with a phosphate group;hydrazone linkages which are reaction product of a hydrazide and analdehyde; acetal linkages that are the reaction product of an aldehydeand an alcohol; orthoester linkages that are the reaction product of aformate and an alcohol; peptide linkages formed by an amine group,including but not limited to, at an end of a polymer such as PEG, and acarboxyl group of a peptide; and oligonucleotide linkages formed by aphosphoramidite group, including but not limited to, at the end of apolymer, and a 5′ hydroxyl group of an oligonucleotide.

The term “biologically active molecule”, “biologically active moiety” or“biologically active agent” when used herein means any substance whichcan affect any physical or biochemical properties of a biologicalsystem, pathway, molecule, or interaction relating to an organism,including but not limited to, viruses, bacteria, bacteriophage,transposon, prion, insects, fungi, plants, animals, and humans. Inparticular, as used herein, biologically active molecules include, butare not limited to, any substance intended for diagnosis, cure,mitigation, treatment, or prevention of disease in humans or otheranimals, or to otherwise enhance physical or mental well-being of humansor animals. Examples of biologically active molecules include, but arenot limited to, peptides, proteins, enzymes, small molecule drugs, harddrugs, soft drugs, carbohydrates, inorganic atoms or molecules, dyes,lipids, nucleosides, radionuclides, oligonucleotides, toxins, cells,viruses, liposomes, microparticles and micelles. Classes of biologicallyactive agents that are suitable for use with the invention include, butare not limited to, drugs, prodrugs, radionuclides, imaging agents,polymers, antibiotics, fungicides, anti-viral agents, anti-inflammatoryagents, anti-tumor agents, cardiovascular agents, anti-anxiety agents,hormones, growth factors, steroidal agents, microbially derived toxins,and the like.

A “bifunctional polymer” refers to a polymer comprising two discretefunctional groups that are capable of reacting specifically with othermoieties (including but not limited to, amino acid side groups) to formcovalent or non-covalent linkages. A bifunctional linker having onefunctional group reactive with a group on a particular biologicallyactive component, and another group reactive with a group on a secondbiological component, may be used to form a conjugate that includes thefirst biologically active component, the bifunctional linker and thesecond biologically active component. Many procedures and linkermolecules for attachment of various compounds to peptides are known.See, e.g., European Patent Application No. 188,256; U.S. Pat. Nos.4,671,958, 4,659,839, 4,414,148, 4,699,784; 4,680,338; and 4,569,789which are incorporated by reference herein. A “multi-functional polymer”refers to a polymer comprising two or more discrete functional groupsthat are capable of reacting specifically with other moieties (includingbut not limited to, amino acid side groups) to form covalent ornon-covalent linkages. A bi-functional polymer or multi-functionalpolymer may be any desired length or molecular weight, and may beselected to provide a particular desired spacing or conformation betweenone or more molecules linked to hGH.

Where substituent groups are specified by their conventional chemicalformulas, written from left to right, they equally encompass thechemically identical substituents that would result from writing thestructure from right to left, for example, the structure —CH₂O— isequivalent to the structure —OCH₂—.

As used herein, the term “modulated serum half-life” means the positiveor negative change in circulating half-life of a modified hGH relativeto its non-modified form. Serum half-life is measured by taking bloodsamples at various time points after administration of hGH, anddetermining the concentration of that molecule in each sample.Correlation of the serum concentration with time allows calculation ofthe serum half-life. Increased serum half-life desirably has at leastabout two-fold, but a smaller increase may be useful, for example whereit enables a satisfactory dosing regimen or avoids a toxic effect. Insome embodiments, the increase is at least about three-fold, at leastabout five-fold, or at least about ten-fold.

The term “modulated therapeutic half-life” as used herein means thepositive or negative change in the half-life of the therapeuticallyeffective amount of hGH polypeptide, relative to its non-modified form.Therapeutic half-life is measured by measuring pharmacokinetic and/orpharmacodynamic properties of the molecule at various time points afteradministration. Increased therapeutic half-life desirably enables aparticular beneficial dosing regimen, a particular beneficial totaldose, or avoids an undesired effect. In some embodiments, the increasedtherapeutic half-life results from increased potency, increased ordecreased binding of the modified molecule to its target, increased ordecreased breakdown of the molecule by enzymes such as proteases, or anincrease or decrease in another parameter or mechanism of action of thenon-modified molecule.

The term “isolated,” when applied to a nucleic acid or protein, denotesthat the nucleic acid or protein is free of at least some of the othercellular components with which it is associated in the natural state, orthat the nucleic acid or protein has been concentrated to a levelgreater than the concentration of its in vivo or in vitro production. Itcan be in a homogeneous state. Isolated substances can be in either adry or semi-dry state, or in solution, including but not limited to, anaqueous solution. It can be a component of a pharmaceutical compositionthat comprises additional pharmaceutically acceptable carriers and/orexcipients. Purity and homogeneity are typically determined usinganalytical chemistry techniques such as polyacrylamide gelelectrophoresis or high performance liquid chromatography. A proteinwhich is the predominant species present in a preparation issubstantially purified. In particular, an isolated gene is separatedfrom open reading frames which flank the gene and encode a protein otherthan the gene of interest. The term “purified” denotes that a nucleicacid or protein gives rise to substantially one band in anelectrophoretic gel. Particularly, it may mean that the nucleic acid orprotein is at least 85% pure, at least 90% pure, at least 95% pure, atleast 99% or greater pure.

The term “nucleic acid” refers to deoxyribonucleotides,deoxyribonucleosides, ribonucleosides, or ribonucleotides and polymersthereof in either single- or double-stranded form. Unless specificallylimited, the term encompasses nucleic acids containing known analoguesof natural nucleotides which have similar binding properties as thereference nucleic acid and are metabolized in a manner similar tonaturally occurring nucleotides. Unless specifically limited otherwise,the term also refers to oligonucleotide analogs including PNA(peptidonucleic acid), analogs of DNA used in antisense technology(phosphorothioates, phosphoroamidates, and the like). Unless otherwiseindicated, a particular nucleic acid sequence also implicitlyencompasses conservatively modified variants thereof (including but notlimited to, degenerate codon substitutions) and complementary sequencesas well as the sequence explicitly indicated. Specifically, degeneratecodon substitutions may be achieved by generating sequences in which thethird position of one or more selected (or all) codons is substitutedwith mixed-base and/or deoxyinosine residues (Batzer et al., NucleicAcid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

The term “amino acid” refers to naturally occurring and non-naturallyoccurring amino acids, as well as amino acid analogs and amino acidmimetics that function in a manner similar to the naturally occurringamino acids. Naturally encoded amino acids are the 20 common amino acids(alanine, arginine, asparagine, aspartic acid, cysteine, glutamine,glutamic acid, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, serine, threonine, tryptophan,tyrosine, and valine) and pyrrolysine and selenocysteine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an α carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, such as,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (such as, norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, “conservatively modified variants” refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of ordinary skill inthe art will recognize that each codon in a nucleic acid (except AUG,which is ordinarily the only codon for methionine, and TGG, which isordinarily the only codon for tryptophan) can be modified to yield afunctionally identical molecule. Accordingly, each silent variation of anucleic acid which encodes a polypeptide is implicit in each describedsequence.

As to amino acid sequences, one of ordinary skill in the art willrecognize that individual substitutions, deletions or additions to anucleic acid, peptide, polypeptide, or protein sequence which alters,adds or deletes a single amino acid or a small percentage of amino acidsin the encoded sequence is a “conservatively modified variant” where thealteration results in the deletion of an amino acid, addition of anamino acid, or substitution of an amino acid with a chemically similaramino acid. Conservative substitution tables providing functionallysimilar amino acids are known to those of ordinary skill in the art.Such conservatively modified variants are in addition to and do notexclude polymorphic variants, interspecies homologs, and alleles of theinvention.

Conservative substitution tables providing functionally similar aminoacids are known to those of ordinary skill in the art. The followingeight groups each contain amino acids that are conservativesubstitutions for one another:

1) Alanine (A), Glycine (G);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

7) Serine (S), Threonine (T); and

8) Cysteine (C), Methionine (M)

(see, e.g., Creighton, Proteins: Structures and Molecular Properties (WH Freeman & Co.; 2nd edition (December 1993)

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same. Sequences are“substantially identical” if they have a percentage of amino acidresidues or nucleotides that are the same (i.e., about 60% identity,about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, orabout 95% identity over a specified region), when compared and alignedfor maximum correspondence over a comparison window, or designatedregion as measured using one of the following sequence comparisonalgorithms (or other algorithms available to persons of ordinary skillin the art) or by manual alignment and visual inspection. Thisdefinition also refers to the complement of a test sequence. Theidentity can exist over a region that is at least about 50 amino acidsor nucleotides in length, or over a region that is 75-100 amino acids ornucleotides in length, or, where not specified, across the entiresequence of a polynucleotide or polypeptide.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are known to those of ordinary skill in the art. Optimalalignment of sequences for comparison can be conducted, including butnot limited to, by the local homology algorithm of Smith and Waterman(1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm ofNeedleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search forsimilarity method of Pearson and Lipman (1988) Proc. Nat'l. Acad. Sci.USA 85:2444, by computerized implementations of these algorithms (GAP,BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package,Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manualalignment and visual inspection (see, e.g., Ausubel et al., CurrentProtocols in Molecular Biology (1995 supplement)).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al. (1997) Nuc. AcidsRes. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410,respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information. TheBLAST algorithm parameters W, T, and X determine the sensitivity andspeed of the alignment. The BLASTN program (for nucleotide sequences)uses as defaults a wordlength (W) of 11, an expectation (E) or 10, M=5,N=−4 and a comparison of both strands. For amino acid sequences, theBLASTP program uses as defaults a wordlength of 3, and expectation (E)of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992)Proc. Natl. Acad. Sci. USA 89:10915) alignments (B) of 50, expectation(E) of 10, M=5, N=−4, and a comparison of both strands. The BLASTalgorithm is typically performed with the “low complexity” filter turnedoff.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid may be considered similar to a referencesequence if the smallest sum probability in a comparison of the testnucleic acid to the reference nucleic acid is less than about 0.2, lessthan about 0.01, or less than about 0.001.

The phrase “selectively (or specifically) hybridizes to” refers to thebinding, duplexing, or hybridizing of a molecule only to a particularnucleotide sequence under stringent hybridization conditions when thatsequence is present in a complex mixture (including but not limited to,total cellular or library DNA or RNA).

The phrase “stringent hybridization conditions” refers to hybridizationof sequences of DNA, RNA, PNA, or other nucleic acid mimics, orcombinations thereof under conditions of low ionic strength and hightemperature as is known in the art. Typically, under stringentconditions a probe will hybridize to its target subsequence in a complexmixture of nucleic acid (including but not limited to, total cellular orlibrary DNA or RNA) but does not hybridize to other sequences in thecomplex mixture. Stringent conditions are sequence-dependent and will bedifferent in different circumstances. Longer sequences hybridizespecifically at higher temperatures. An extensive guide to thehybridization of nucleic acids is found in Tijssen, LaboratoryTechniques in Biochemistry and Molecular Biology—Hybridization withNucleic Probes, “Overview of principles of hybridization and thestrategy of nucleic acid assays” (1993). Generally, stringent conditionsare selected to be about 5-10° C. lower than the thermal melting point(T_(m)) for the specific sequence at a defined ionic strength pH. TheT_(m) is the temperature (under defined ionic strength, pH, and nucleicconcentration) at which 50% of the probes complementary to the targethybridize to the target sequence at equilibrium (as the target sequencesare present in excess, at T_(m), 50% of the probes are occupied atequilibrium). Stringent conditions may be those in which the saltconcentration is less than about 1.0 M sodium ion, typically about 0.01to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 andthe temperature is at least about 30° C. for short probes (including butnot limited to, 10 to 50 nucleotides) and at least about 60° C. for longprobes (including but not limited to, greater than 50 nucleotides).Stringent conditions may also be achieved with the addition ofdestabilizing agents such as formamide. For selective or specifichybridization, a positive signal may be at least two times background,optionally 10 times background hybridization. Exemplary stringenthybridization conditions can be as following: 50% formamide, 5×SSC, and1% SDS, incubating at 42° C., or 5×SSC, 1% SDS, incubating at 65° C.,with wash in 0.2×SSC, and 0.1% SDS at 65° C. Such washes can beperformed for 5, 15, 30, 60, 120, or more minutes.

As used herein, the term “eukaryote” refers to organisms belonging tothe phylogenetic domain Eucarya such as animals (including but notlimited to, mammals, insects, reptiles, birds, etc.), ciliates, plants(including but not limited to, monocots, dicots, algae, etc.), fungi,yeasts, flagellates, microsporidia, protists, etc.

As used herein, the term “non-eukaryote” refers to non-eukaryoticorganisms. For example, a non-eukaryotic organism can belong to theEubacteria (including but not limited to, Escherichia coli, Thermusthermophilus, Bacillus stearothermophilus, Pseudomonas fluorescens,Pseudomonas aeruginosa, Pseudomonas putida, etc.) phylogenetic domain,or the Archaea (including but not limited to, Methanococcus jannaschii,Methanobacterium thermoautotrophicum, Halobacterium such as Haloferaxvolcanii and Halobacterium species NRC-1, Archaeoglobus fulgidus,Pyrococcus furiosus, Pyrococcus horikoshii, Aeuropyrum pernix, etc.)phylogenetic domain.

The term “subject” as used herein, refers to an animal, in someembodiments a mammal, and in other embodiments a human, who is theobject of treatment, observation or experiment.

The term “effective amount” as used herein refers to that amount of thenon-natural amino acid polypeptide being administered which will relieveto some extent one or more of the symptoms of the disease, condition ordisorder being treated. Compositions containing the non-natural aminoacid polypeptide described herein can be administered for prophylactic,enhancing, and/or therapeutic treatments.

The terms “enhance” or “enhancing” means to increase or prolong eitherin potency or duration a desired effect. Thus, in regard to enhancingthe effect of therapeutic agents, the term “enhancing” refers to theability to increase or prolong, either in potency or duration, theeffect of other therapeutic agents on a system. An “enhancing-effectiveamount,” as used herein, refers to an amount adequate to enhance theeffect of another therapeutic agent in a desired system. When used in apatient, amounts effective for this use will depend on the severity andcourse of the disease, disorder or condition, previous therapy, thepatient's health status and response to the drugs, and the judgment ofthe treating physician.

The term “modified,” as used herein refers to any changes made to agiven polypeptide, such as changes to the length of the polypeptide, theamino acid sequence, chemical structure, co-translational modification,or post-translational modification of a polypeptide. The form“(modified)” term means that the polypeptides being discussed areoptionally modified, that is, the polypeptides under discussion can bemodified or unmodified.

The term “post-translationally modified” refers to any modification of anatural or non-natural amino acid that occurs to such an amino acidafter it has been incorporated into a polypeptide chain. The termencompasses, by way of example only, co-translational in vivomodifications, co-translational in vitro modifications (such as in acell-free translation system), post-translational in vivo modifications,and post-translational in vitro modifications.

In prophylactic applications, compositions containing the non-naturalamino acid polypeptide are administered to a patient susceptible to orotherwise at risk of a particular disease, disorder or condition. Suchan amount is defined to be a “prophylactically effective amount.” Inthis use, the precise amounts also depend on the patient's state ofhealth, weight, and the like. It is considered well within the skill ofthe art for one to determine such prophylactically effective amounts byroutine experimentation (e.g., a dose escalation clinical trial).

The term “protected” refers to the presence of a “protecting group” ormoiety that prevents reaction of the chemically reactive functionalgroup under certain reaction conditions. The protecting group will varydepending on the type of chemically reactive group being protected. Forexample, if the chemically reactive group is an amine or a hydrazide,the protecting group can be selected from the group oftert-butyloxycarbonyl (t-Boc) and 9-fluorenylmethoxycarbonyl (Fmoc). Ifthe chemically reactive group is a thiol, the protecting group can beorthopyridyldisulfide. If the chemically reactive group is a carboxylicacid, such as butanoic or propionic acid, or a hydroxyl group, theprotecting group can be benzyl or an alkyl group such as methyl, ethyl,or tert-butyl. Other protecting groups known in the art may also be usedin or with the methods and compositions described herein, includingphotolabile groups such as Nvoc and MeNvoc. Other protecting groupsknown in the art may also be used in or with the methods andcompositions described herein.

By way of example only, blocking/protecting groups may be selected from:

Other protecting groups are described in Greene and Wuts, ProtectiveGroups in Organic Synthesis, 3rd Ed., John Wiley & Sons, New York, N.Y.,1999, which is incorporated herein by reference in its entirety.

In therapeutic applications, compositions containing the (modified)non-natural amino acid polypeptide are administered to a patient alreadysuffering from a disease, condition or disorder, in an amount sufficientto cure or at least partially arrest the symptoms of the disease,disorder or condition. Such an amount is defined to be a“therapeutically effective amount,” and will depend on the severity andcourse of the disease, disorder or condition, previous therapy, thepatient's health status and response to the drugs, and the judgment ofthe treating physician. It is considered well within the skill of theart for one to determine such therapeutically effective amounts byroutine experimentation (e.g., a dose escalation clinical trial).

The term “treating” is used to refer to either prophylactic and/ortherapeutic treatments.

Non-naturally encoded amino acid polypeptides presented herein mayinclude isotopically-labelled compounds with one or more atoms replacedby an atom having an atomic mass or mass number different from theatomic mass or mass number usually found in nature. Examples of isotopesthat can be incorporated into the present compounds include isotopes ofhydrogen, carbon, nitrogen, oxygen, fluorine and chlorine, such as ²H,³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, ³⁵S, ¹⁸F, ³⁶Cl, respectively. Certainisotopically-labelled compounds described herein, for example those intowhich radioactive isotopes such as ³H and ¹⁴C are incorporated, may beuseful in drug and/or substrate tissue distribution assays. Further,substitution with isotopes such as deuterium, i.e., ²H, can affordcertain therapeutic advantages resulting from greater metabolicstability, for example increased in vivo half-life or reduced dosagerequirements.

All isomers including but not limited to diastereomers, enantiomers, andmixtures thereof are considered as part of the compositions describedherein. In additional or further embodiments, the non-naturally encodedamino acid polypeptides are metabolized upon administration to anorganism in need to produce a metabolite that is then used to produce adesired effect, including a desired therapeutic effect. In further oradditional embodiments are active metabolites of non-naturally encodedamino acid polypeptides.

In some situations, non-naturally encoded amino acid polypeptides mayexist as tautomers. In addition, the non-naturally encoded amino acidpolypeptides described herein can exist in unsolvated as well assolvated forms with pharmaceutically acceptable solvents such as water,ethanol, and the like. The solvated forms are also considered to bedisclosed herein. Those of ordinary skill in the art will recognize thatsome of the compounds herein can exist in several tautomeric forms. Allsuch tautomeric forms are considered as part of the compositionsdescribed herein.

DETAILED DESCRIPTION

I. Introduction

hGH molecules comprising at least one unnatural amino acid are providedin the invention. In certain embodiments of the invention, the hGHpolypeptide with at least one unnatural amino acid includes at least onepost-translational modification. In one embodiment, the at least onepost-translational modification comprises attachment of a moleculeincluding but not limited to, a label, a dye, a polymer, a water-solublepolymer, a derivative of polyethylene glycol, a photocrosslinker, aradionuclide, a cytotoxic compound, a drug, an affinity label, aphotoaffinity label, a reactive compound, a resin, a second protein orpolypeptide or polypeptide analog, an antibody or antibody fragment, ametal chelator, a cofactor, a fatty acid, a carbohydrate, apolynucleotide, a DNA, a RNA, an antisense polynucleotide, a saccharide,a water-soluble dendrimer, a cyclodextrin, an inhibitory ribonucleicacid, a biomaterial, a nanoparticle, a spin label, a fluorophore, ametal-containing moiety, a radioactive moiety, a novel functional group,a group that covalently or noncovalently interacts with other molecules,a photocaged moiety, an actinic radiation excitable moiety, aphotoisomerizable moiety, biotin, a derivative of biotin, a biotinanalogue, a moiety incorporating a heavy atom, a chemically cleavablegroup, a photocleavable group, an elongated side chain, a carbon-linkedsugar, a redox-active agent, an amino thioacid, a toxic moiety, anisotopically labeled moiety, a biophysical probe, a phosphorescentgroup, a chemiluminescent group, an electron dense group, a magneticgroup, an intercalating group, a chromophore, an energy transfer agent,a biologically active agent, a detectable label, a small molecule, aquantum dot, a nanotransmitter, a radionucleotide, a radiotransmitter, aneutron-capture agent, or any combination of the above or any otherdesirable compound or substance, comprising a second reactive group toat least one unnatural amino acid comprising a first reactive grouputilizing chemistry methodology that is known to one of ordinary skillin the art to be suitable for the particular reactive groups. In certainembodiments of the modified hGH polypeptide of the present invention, atleast one unnatural amino acid (including but not limited to, unnaturalamino acid containing a keto functional group) comprising at least onepost-translational modification, is used where the at least onepost-translational modification comprises a saccharide moiety. Incertain embodiments, the post-translational modification is made in vivoin a eukaryotic cell or in a non-eukaryotic cell.

In certain embodiments, the protein includes at least onepost-translational modification that is made in vivo by one host cell,where the post-translational modification is not normally made byanother host cell type. In certain embodiments, the protein includes atleast one post-translational modification that is made in vivo by aeukaryotic cell, where the post-translational modification is notnormally made by a non-eukaryotic cell. Examples of post-translationalmodifications include, but are not limited to, glycosylation,acetylation, acylation, lipid-modification, palmitoylation, palmitateaddition, phosphorylation, glycolipid-linkage modification, and thelike. In one embodiment, the post-translational modification comprisesattachment of an oligosaccharide to an asparagine by a GlcNAc-asparaginelinkage (including but not limited to, where the oligosaccharidecomprises (GlcNAc-Man)₂-Man-GlcNAc-GlcNAc, and the like). In anotherembodiment, the post-translational modification comprises attachment ofan oligosaccharide (including but not limited to, Gal-GalNAc,Gal-GlcNAc, etc.) to a serine or threonine by a GalNAc-serine, aGalNAc-threonine, a GlcNAc-serine, or a GlcNAc-threonine linkage. Incertain embodiments, a protein or polypeptide of the invention cancomprise a secretion or localization sequence, an epitope tag, a FLAGtag, a polyhistidine tag, a GST fusion, and/or the like. Examples ofsecretion signal sequences include, but are not limited to, aprokaryotic secretion signal sequence, a eukaryotic secretion signalsequence, a eukaryotic secretion signal sequence 5′-optimized forbacterial expression, a novel secretion signal sequence, pectate lyasesecretion signal sequence, Omp A secretion signal sequence, and a phagesecretion signal sequence. Examples of secretion signal sequences,include, but are not limited to, STII (prokaryotic), Fd GIII and M13(phage), Bgl2 (yeast), and the signal sequence bla derived from atransposon.

The protein or polypeptide of interest can contain at least one, atleast two, at least three, at least four, at least five, at least six,at least seven, at least eight, at least nine, or ten or more unnaturalamino acids. The unnatural amino acids can be the same or different, forexample, there can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more differentsites in the protein that comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or moredifferent unnatural amino acids. In certain embodiments, at least one,but fewer than all, of a particular amino acid present in a naturallyoccurring version of the protein is substituted with an unnatural aminoacid.

The present invention provides methods and compositions based on growthhormone, in particular hGH, comprising at least one non-naturallyencoded amino acid. Introduction of at least one non-naturally encodedamino acid into hGH can allow for the application of conjugationchemistries that involve specific chemical reactions, including, but notlimited to, with one or more non-naturally encoded amino acids while notreacting with the commonly occurring 20 amino acids. In someembodiments, hGH comprising the non-naturally encoded amino acid islinked or bonded to a water soluble polymer, such as polyethylene glycol(PEG), via the side chain of the non-naturally encoded amino acid. Thisinvention provides a highly efficient method for the selectivemodification of proteins with PEG derivatives, which involves theselective incorporation of non-genetically encoded amino acids,including but not limited to, those amino acids containing functionalgroups or substituents not found in the 20 naturally incorporated aminoacids, including but not limited to a ketone moiety, into proteins inresponse to a selector codon and the subsequent modification of thoseamino acids with a suitably reactive PEG derivative. Once incorporated,the amino acid side chains can then be modified by utilizing chemistrymethodologies known to those of ordinary skill in the art to be suitablefor the particular functional groups or substituents present in thenon-naturally encoded amino acid. Known chemistry methodologies of awide variety are suitable for use in the present invention toincorporate a water soluble polymer into the protein.

The present invention provides conjugates of hGH polypeptide having awide variety of functional groups, substituents or moieties, with othersubstances including but not limited to a label; a dye; a polymer; awater-soluble polymer; a derivative of polyethylene glycol; aphotocrosslinker; a radionuclide; a cytotoxic compound; a drug; anaffinity label; a photoaffinity label; a reactive compound; a resin; asecond protein or polypeptide or polypeptide analog; an antibody orantibody fragment; a metal chelator; a cofactor; a fatty acid; acarbohydrate; a polynucleotide; a DNA; a RNA; an antisensepolynucleotide; a saccharide; a water-soluble dendrimer; a cyclodextrin;an inhibitory ribonucleic acid; a biomaterial; a nanoparticle; a spinlabel; a fluorophore, a metal-containing moiety; a radioactive moiety; anovel functional group; a group that covalently or noncovalentlyinteracts with other molecules; a photocaged moiety; an actinicradiation excitable moiety; a photoisomerizable moiety; biotin; aderivative of biotin; a biotin analogue; a moiety incorporating a heavyatom; a chemically cleavable group; a photocleavable group; an elongatedside chain; a carbon-linked sugar; a redox-active agent; an aminothioacid; a toxic moiety; an isotopically labeled moiety; a biophysicalprobe; a phosphorescent group; a chemiluminescent group; an electrondense group; a magnetic group; an intercalating group; a chromophore; anenergy transfer agent; a biologically active agent; a detectable label;a small molecule; a quantum dot; a nanotransmitter; a radionuclide; aradiotransmitter; a neutron-capture agent; or any combination of theabove, or any other desirable compound or substance.

It is well established in the art that PEG can be used to modify thesurfaces of biomaterials (see, e.g., U.S. Pat. No. 6,610,281; Mehvar,R., J. Pharm Pharm Sci., 3(1):125-136 (2000) which are incorporated byreference herein). The PEG derivative can be bonded directly to thepolymer via a reactive moiety. Alternatively, the PEG derivative can beprepared by attaching a linking agent that has a reactive moiety at oneterminus to a conventional activated polymer so that the resultingpolymer has the reactive moiety at its terminus. Alternatively, a watersoluble polymer having at least one active nucleophilic or electrophilicmoiety undergoes a reaction with a linking agent that has a reactivegroup at one terminus so that a covalent bond is formed between the PEGpolymer and the linking agent and the reactive group is positioned atthe terminus of the polymer. Nucleophilic and electrophilic moieties,including amines, thiols, hydrazides, hydrazines, alcohols,carboxylates, aldehydes, ketones, thioesters and the like, are known tothose of ordinary skill in the art. The PEG derivatives can be used tomodify the properties of surfaces and molecules where biocompatibility,stability, solubility and lack of immunogenicity are important, while atthe same time providing a more selective means of attaching the PEGderivatives to proteins than was previously known in the art.

II. Growth Hormone Supergene Family

The following proteins include those encoded by genes of the growthhormone (GH) supergene family (Bazan, F., Immunology Today 11: 350-354(1990); Bazan, J. F. Science 257: 410-413 (1992); Mott, H. R. andCampbell, I. D., Current Opinion in Structural Biology 5: 114-121(1995); Silvennoinen, O. and Ihle, J. N., SIGNALLING BY THEHEMATOPOIETIC CYTOKINE RECEPTORS (1996)): growth hormone, prolactin,placental lactogen, erythropoietin (EPO), thrombopoietin (TPO),interleukin-2 (IL-2), IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11,IL-12 (p35 subunit), IL-13, IL-15, oncostatin M, ciliary neurotrophicfactor (CNTF), leukemia inhibitory factor (LIF), alpha interferon, betainterferon, epsilon interferon, gamma interferon, omega interferon, tauinterferon, granulocyte-colony stimulating factor (G-CSF),granulocyte-macrophage colony stimulating factor (GM-CSF), macrophagecolony stimulating factor (M-CSF) and cardiotrophin-1 (CT-1) (“the GHsupergene family”). It is anticipated that additional members of thisgene family will be identified in the future through gene cloning andsequencing. Members of the GH supergene family have similar secondaryand tertiary structures, despite the fact that they generally havelimited amino acid or DNA sequence identity. The shared structuralfeatures allow new members of the gene family to be readily identifiedand the non-natural amino acid methods and compositions described hereinsimilarly applied. Given the extent of structural homology among themembers of the GH supergene family, non-naturally encoded amino acidsmay be incorporated into any members of the GH supergene family usingthe present invention. Each member of this family of proteins comprisesa four helical bundle.

Structures of a number of cytokines, including G-CSF (Zink et al., FEBSLett. 314:435 (1992); Zink et al., Biochemistry 33:8453 (1994); Hill etal., Proc. Natl. Acad. Sci. USA 90:5167 (1993)), GM-CSF (Diederichs, K.,et al. Science 154: 1779-1782 (1991); Walter et al., J. Mol. Biol.224:1075-1085 (1992)), IL-2 (Bazan, J. F. and McKay, D. B. Science 257:410-413 (1992), IL-4 (Redfield et al., Biochemistry 30: 11029-11035(1991); Powers et al., Science 256:1673-1677 (1992)), and IL-5 (Milburnet al., Nature 363: 172-176 (1993)) have been determined by X-raydiffraction and NMR studies and show striking conservation with the GHstructure, despite a lack of significant primary sequence homology. IFNis considered to be a member of this family based upon modeling andother studies (Lee et al., J. Interferon Cytokine Res. 15:341 (1995);Murgolo et al., Proteins 17:62 (1993); Radhakrishnan et al., Structure4:1453 (1996); Klaus et al., J. Mol. Biol. 274:661 (1997)). EPO isconsidered to be a member of this family based upon modeling andmutagenesis studies (Boissel et al., J. Biol. Chem. 268: 15983-15993(1993); Wen et al., J. Biol. Chem. 269: 22839-22846 (1994)). All of theabove cytokines and growth factors are now considered to comprise onelarge gene family.

In addition to sharing similar secondary and tertiary structures,members of this family share the property that they must oligomerizecell surface receptors to activate intracellular signaling pathways.Some GH family members, including but not limited to; GH and EPO, bind asingle type of receptor and cause it to form homodimers. Other familymembers, including but not limited to, IL-2, IL-4, and IL-6, bind morethan one type of receptor and cause the receptors to form heterodimersor higher order aggregates (Davis et al., (1993), Science 260:1805-1808; Paonessa et al., (1995), EMBO J. 14: 1942-1951; Mott andCampbell, Current Opinion in Structural Biology 5: 114-121 (1995)).Mutagenesis studies have shown that, like GH, these other cytokines andgrowth factors contain multiple receptor binding sites, typically two,and bind their cognate receptors sequentially (Mott and Campbell,Current Opinion in Structural Biology 5: 114-121 (1995); Matthews etal., (1996) Proc. Natl. Acad. Sci. USA 93: 9471-9476). Like GH, theprimary receptor binding sites for these other family members occurprimarily in the four alpha helices and the A-B loop. The specific aminoacids in the helical bundles that participate in receptor binding differamongst the family members. Most of the cell surface receptors thatinteract with members of the GH supergene family are structurallyrelated and comprise a second large multi-gene family. See, e.g. U.S.Pat. No. 6,608,183, which is incorporated by reference herein.

A general conclusion reached from mutational studies of various membersof the GH supergene family is that the loops joining the alpha helicesgenerally tend to not be involved in receptor binding. In particular theshort B-C loop appears to be non-essential for receptor binding in most,if not all, family members. For this reason, the B-C loop may besubstituted with non-naturally encoded amino acids as described hereinin members of the GH supergene family. The A-B loop, the C-D loop (andD-E loop of interferon/IL-10-like members of the GH superfamily) mayalso be substituted with a non-naturally-occurring amino acid. Aminoacids proximal to helix A and distal to the final helix also tend not tobe involved in receptor binding and also may be sites for introducingnon-naturally-occurring amino acids. In some embodiments, anon-naturally encoded amino acid is substituted at any position within aloop structure, including but not limited to, the first 1, 2, 3, 4, 5,6, 7, or more amino acids of the A-B, B-C, C-D or D-E loop. In someembodiments, one or more non-naturally encoded amino acids aresubstituted within the last 1, 2, 3, 4, 5, 6, 7, or more amino acids ofthe A-B, B-C, C-D or D-E loop.

Certain members of the GH family, including but not limited to, EPO,IL-2, IL-3, IL-4, IL-6, G-CSF, GM-CSF, TPO, IL-1, IL-12 p35, IL-13,IL-15 and beta interferon contain N-linked and/or O-linked sugars. Theglycosylation sites in the proteins occur almost exclusively in the loopregions and not in the alpha helical bundles. Because the loop regionsgenerally are not involved in receptor binding and because they aresites for the covalent attachment of sugar groups, they may be usefulsites for introducing non-naturally-occurring amino acid substitutionsinto the proteins. Amino acids that comprise the N- and O-linkedglycosylation sites in the proteins may be sites fornon-naturally-occurring amino acid substitutions because these aminoacids are surface-exposed. Therefore, the natural protein can toleratebulky sugar groups attached to the proteins at these sites and theglycosylation sites tend to be located away from the receptor bindingsites.

Additional members of the GH supergene family are likely to bediscovered in the future. New members of the GH supergene family can beidentified through computer-aided secondary and tertiary structureanalyses of the predicted protein sequences, and by selection techniquesdesigned to identify molecules that bind to a particular target. Membersof the GH supergene family typically possess four or five amphipathichelices joined by non-helical amino acids (the loop regions). Theproteins may contain a hydrophobic signal sequence at their N-terminusto promote secretion from the cell. Such later discovered members of theGH supergene family also are included within this invention. A relatedapplication is International Patent Application entitled “Modified FourHelical Bundle Polypeptides and Their Uses” published as WO 05/074650 onAug. 18, 2005, which is incorporated by reference herein.

Thus, the description of the hGH is provided for illustrative purposesand by way of example only and not as a limit on the scope of themethods, compositions, strategies and techniques described herein.Further, reference to hGH polypeptides in this application is intendedto use the generic term as an example of any growth hormone. Thus, it isunderstood that the modifications and chemistries described herein withreference to hGH polypeptides or protein can be equally applied to anymember of the GH supergene family, including those specifically listedherein.

III. Selector Codons

Selector codons of the invention expand the genetic codon framework ofprotein biosynthetic machinery. For example, a selector codon includes,but is not limited to, a unique three base codon, a nonsense codon, suchas a stop codon, including but not limited to, an amber codon (UAG), anochre codon, or an opal codon (UGA), an unnatural codon, a four or morebase codon, a rare codon, or the like. It is readily apparent to thoseof ordinary skill in the art that there is a wide range in the number ofselector codons that can be introduced into a desired gene orpolynucleotide, including but not limited to, one or more, two or more,three or more, 4, 5, 6, 7, 8, 9, 10 or more in a single polynucleotideencoding at least a portion of the hGH polypeptide.

In one embodiment, the methods involve the use of a selector codon thatis a stop codon for the incorporation of one or more unnatural aminoacids in vivo. For example, an O-tRNA is produced that recognizes thestop codon, including but not limited to, UAG, and is aminoacylated byan O-RS with a desired unnatural amino acid. This O-tRNA is notrecognized by the naturally occurring host's aminoacyl-tRNA synthetases.Conventional site-directed mutagenesis can be used to introduce the stopcodon, including but, not limited to, TAG, at the site of interest in apolypeptide of interest. See, e.g., Sayers, J. R., et al. (1988), 5′-3′Exonucleases in phosphorothioate-based oligonucleotide-directedmutagenesis. Nucleic Acids Res, 16:791-802. When the O-RS, O-tRNA andthe nucleic acid that encodes the polypeptide of interest are combinedin vivo, the unnatural amino acid is incorporated in response to the UAGcodon to give a polypeptide containing the unnatural amino acid at thespecified position.

The incorporation of unnatural amino acids in vivo can be done withoutsignificant perturbation of the eukaryotic host cell. For example,because the suppression efficiency for the UAG codon depends upon thecompetition between the O-tRNA, including but not limited to, the ambersuppressor tRNA, and a eukaryotic release factor (including but notlimited to, eRF) (which binds to a stop codon and initiates release ofthe growing peptide from the ribosome), the suppression efficiency canbe modulated by, including but not limited to, increasing the expressionlevel of O-tRNA, and/or the suppressor tRNA.

Unnatural amino acids can also be encoded with rare codons. For example,when the arginine concentration in an in vitro protein synthesisreaction is reduced, the rare arginine codon, AGG, has proven to beefficient for insertion of Ala by a synthetic tRNA acylated withalanine. See, e.g., Ma et al., Biochemistry, 32:7939 (1993). In thiscase, the synthetic tRNA competes with the naturally occurring tRNAArg,which exists as a minor species in Escherichia coli. Some organisms donot use all triplet codons. An unassigned codon AGA in Micrococcusluteus has been utilized for insertion of amino acids in an in vitrotranscription/translation extract. See, e.g., Kowal and Oliver, Nucl.Acid. Res., 25:4685 (1997). Components of the present invention can begenerated to use these rare codons in vivo.

Selector codons also comprise extended codons, including but not limitedto, four or more base codons, such as, four, five, six or more basecodons. Examples of four base codons include, including but are notlimited to, AGGA, CUAG, UAGA, CCCU and the like. Examples of five basecodons include, but are not limited to, AGGAC, CCCCU, CCCUC, CUAGA,CUACU, UAGGC and the like. A feature of the invention includes usingextended codons based on frameshift suppression. Four or more basecodons can insert, including but not limited to, one or multipleunnatural amino acids into the same protein. For example, in thepresence of mutated O-tRNAs, including but not limited to, a specialframeshift suppressor tRNAs, with anticodon loops, for example, with atleast 8-10 nt anticodon loops, the four or more base codon is read assingle amino acid. In other embodiments, the anticodon loops can decode,including but not limited to, at least a four-base codon, at least afive-base codon, or at least a six-base codon or more. Since there are256 possible four-base codons, multiple unnatural amino acids can beencoded in the same cell using a four or more base codon. See, Andersonet al., (2002) Exploring the Limits of Codon and Anticodon Size,Chemistry and Biology, 9:237-244; Magliery, (2001) Expanding the GeneticCode: Selection of Efficient Suppressors of Four-base Codons andIdentification of “Shifty” Four-base Codons with a Library Approach inEscherichia coli, J. Mol. Biol. 307: 755-769.

For example, four-base codons have been used to incorporate unnaturalamino acids into proteins using in vitro biosynthetic methods. See,e.g., Ma et al., (1993) Biochemistry, 32:7939; and Hohsaka et al.,(1999) J. Am. Chem. Soc., 121:34. CGGG and AGGU were used tosimultaneously incorporate 2-naphthylalanine and an NBD derivative oflysine into streptavidin in vitro with two chemically acylatedframeshift suppressor tRNAs. See, e.g., Hohsaka et al., (1999) J. Am.Chem. Soc., 121:12194. In an in vivo study, Moore et al. examined theability of tRNALeu derivatives with NCUA anticodons to suppress UAGNcodons (N can be U, A, G, or C), and found that the quadruplet UAGA canbe decoded by a tRNALeu with a UCUA anticodon with an efficiency of 13to 26% with little decoding in the 0 or −1 frame. See, Moore et al.,(2000) J. Mol. Biol., 298:195. In one embodiment, extended codons basedon rare codons or nonsense codons can be used in the present invention,which can reduce missense read through and frameshift suppression atother unwanted sites.

For a given system, a selector codon can also include one of the naturalthree base codons, where the endogenous system does not use (or rarelyuses) the natural base codon. For example, this includes a system thatis lacking a tRNA that recognizes the natural three base codon, and/or asystem where the three base codon is a rare codon.

Selector codons optionally include unnatural base pairs. These unnaturalbase pairs further expand the existing genetic alphabet. One extra basepair increases the number of triplet codons from 64 to 125. Propertiesof third base pairs include stable and selective base pairing, efficientenzymatic incorporation into DNA with high fidelity by a polymerase, andthe efficient continued primer extension after synthesis of the nascentunnatural base pair. Descriptions of unnatural base pairs which can beadapted for methods and compositions include, e.g., Hirao, et al.,(2002) An unnatural base pair for incorporating amino acid analoguesinto protein, Nature Biotechnology, 20:177-182. See, also, Wu, Y., etal., (2002) J. Am. Chem. Soc. 124:14626-14630. Other relevantpublications are listed below.

For in vivo usage, the unnatural nucleoside is membrane permeable and isphosphorylated to form the corresponding triphosphate. In addition, theincreased genetic information is stable and not destroyed by cellularenzymes. Previous efforts by Benner and others took advantage ofhydrogen bonding patterns that are different from those in canonicalWatson-Crick pairs, the most noteworthy example of which is theiso-C:iso-G pair. See, e.g., Switzer et al., (1989) J. Am. Chem. Soc.,111:8322; and Piccirilli et al., (1990) Nature, 343:33; Kool, (2000)Curr. Opin. Chem. Biol., 4:602. These bases in general mispair to somedegree with natural bases and cannot be enzymatically replicated. Kooland co-workers demonstrated that hydrophobic packing interactionsbetween bases can replace hydrogen bonding to drive the formation ofbase pair. See, Kool, (2000) Curr. Opin. Chem. Biol., 4:602; and Guckianand Kool, (1998) Angew. Chem. Int. Ed. Engl., 36, 2825. In an effort todevelop an unnatural base pair satisfying all the above requirements,Schultz, Romesberg and co-workers have systematically synthesized andstudied a series of unnatural hydrophobic bases. A PICS:PICS self-pairis found to be more stable than natural base pairs, and can beefficiently incorporated into DNA by Klenow fragment of Escherichia coliDNA polymerase I (KF). See, e.g., McMinn et al., (1999) J. Am. Chem.Soc., 121:11585-6; and Ogawa et al., (2000) J. Am. Chem. Soc., 122:3274.A 3MN:3MN self-pair can be synthesized by KF with efficiency andselectivity sufficient for biological function. See, e.g., Ogawa et al.,(2000) J. Am. Chem. Soc., 122:8803. However, both bases act as a chainterminator for further replication. A mutant DNA polymerase has beenrecently evolved that can be used to replicate the PICS self pair. Inaddition, a 7AI self pair can be replicated. See, e.g., Tae et al.,(2001) J. Am. Chem. Soc., 123:7439. A novel metallobase pair, Dipic:Py,has also been developed, which forms a stable pair upon binding Cu(II).See, Meggers et al., (2000) J. Am. Chem. Soc., 122:10714. Becauseextended codons and unnatural codons are intrinsically orthogonal tonatural codons, the methods of the invention can take advantage of thisproperty to generate orthogonal tRNAs for them.

A translational bypassing system can also be used to incorporate anunnatural amino acid in a desired polypeptide. In a translationalbypassing system, a large sequence is incorporated into a gene but isnot translated into protein. The sequence contains a structure thatserves as a cue to induce the ribosome to hop over the sequence andresume translation downstream of the insertion.

In certain embodiments, the protein or polypeptide of interest (orportion thereof) in the methods and/or compositions of the invention isencoded by a nucleic acid. Typically, the nucleic acid comprises atleast one selector codon, at least two selector codons, at least threeselector codons, at least four selector codons, at least five selectorcodons, at least six selector codons, at least seven selector codons, atleast eight selector codons, at least nine selector codons, ten or moreselector codons.

Genes coding for proteins or polypeptides of interest can be mutagenizedusing methods well-known to one of skill in the art and described hereinto include, for example, one or more selector codon for theincorporation of an unnatural amino acid. For example, a nucleic acidfor a protein of interest is mutagenized to include one or more selectorcodon, providing for the incorporation of one or more unnatural aminoacids. The invention includes any such variant, including but notlimited to, mutant, versions of any protein, for example, including atleast one unnatural amino acid. Similarly, the invention also includescorresponding nucleic acids, i.e., any nucleic acid with one or moreselector codon that encodes one or more unnatural amino acid.

Nucleic acid molecules encoding a protein of interest such as a hGHpolypeptide may be readily mutated to introduce a cysteine at anydesired position of the polypeptide. Cysteine is widely used tointroduce reactive molecules, water soluble polymers, proteins, or awide variety of other molecules, onto a protein of interest. Methodssuitable for the incorporation of cysteine into a desired position of apolypeptide are known to those of ordinary skill in the art, such asthose described in U.S. Pat. No. 6,608,183, which is incorporated byreference herein, and standard mutagenesis techniques.

IV. Non-Naturally Encoded Amino Acids

A very wide variety of non-naturally encoded amino acids are suitablefor use in the present invention. Any number of non-naturally encodedamino acids can be introduced into a hGH polypeptide. In general, theintroduced non-naturally encoded amino acids are substantiallychemically inert toward the 20 common, genetically-encoded amino acids(i.e., alanine, arginine, asparagine, aspartic acid, cysteine,glutamine, glutamic acid, glycine, histidine, isoleucine, leucine,lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, and valine). In some embodiments, thenon-naturally encoded amino acids include side chain functional groupsthat react efficiently and selectively with functional groups not foundin the 20 common amino acids (including but not limited to, azido,ketone, aldehyde and aminooxy groups) to form stable conjugates.

The generic structure of an alpha-amino acid is illustrated as follows(Formula I):

A non-naturally encoded amino acid is typically any structure having theabove-listed formula wherein the R group is any substituent other thanone used in the twenty natural amino acids, and may be suitable for usein the present invention. Because the non-naturally encoded amino acidsof the invention typically differ from the natural amino acids only inthe structure of the side chain, the non-naturally encoded amino acidsform amide bonds with other amino acids, including but not limited to,natural or non-naturally encoded, in the same manner in which they areformed in naturally occurring polypeptides. However, the non-naturallyencoded amino acids have side chain groups that distinguish them fromthe natural amino acids. For example, R optionally comprises an alkyl-,aryl-, acyl-, keto-, azido-, hydroxyl-, hydrazine, cyano-, halo-,hydrazide, alkenyl, alkynl, ether, thiol, seleno-, sulfonyl-, borate,boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine,aldehyde, ester, thioacid, hydroxylamine, amino group, or the like orany combination thereof. Other non-naturally occurring amino acids ofinterest that may be suitable for use in the present invention include,but are not limited to, amino acids comprising a photoactivatablecross-linker, spin-labeled amino acids, fluorescent amino acids, metalbinding amino acids, metal-containing amino acids, radioactive aminoacids, amino acids with novel functional groups, amino acids thatcovalently or noncovalently interact with other molecules, photocagedand/or photoisomerizable amino acids, amino acids comprising biotin or abiotin analogue, glycosylated amino acids such as a sugar substitutedserine, other carbohydrate modified amino acids, keto-containing aminoacids, amino acids comprising polyethylene glycol or polyether, heavyatom substituted amino acids, chemically cleavable and/or photocleavableamino acids, amino acids with an elongated side chains as compared tonatural amino acids, including but not limited to, polyethers or longchain hydrocarbons, including but not limited to, greater than about 5or greater than about 10 carbons, carbon-linked sugar-containing aminoacids, redox-active amino acids, amino thioacid containing amino acids,and amino acids comprising one or more toxic moiety.

Exemplary non-naturally encoded amino acids that may be suitable for usein the present invention and that are useful for reactions with watersoluble polymers include, but are not limited to, those with carbonyl,aminooxy, hydrazine, hydrazide, semicarbazide, azide and alkyne reactivegroups. In some embodiments, non-naturally encoded amino acids comprisea saccharide moiety. Examples of such amino acids includeN-acetyl-L-glucosaminyl-L-serine, N-acetyl-L-galactosaminyl-L-serine,N-acetyl-L-glucosaminyl-L-threonine,N-acetyl-L-glucosaminyl-L-asparagine and O-mannosaminyl-L-serine.Examples of such amino acids also include examples where thenaturally-occurring N- or O-linkage between the amino acid and thesaccharide is replaced by a covalent linkage not commonly found innature—including but not limited to, an alkene, an oxime, a thioether,an amide and the like. Examples of such amino acids also includesaccharides that are not commonly found in naturally-occurring proteinssuch as 2-deoxy-glucose, 2-deoxygalactose and the like.

Many of the non-naturally encoded amino acids provided herein arecommercially available, e.g., from Sigma-Aldrich (St. Louis, Mo., USA),Novabiochem (a division of EMD Biosciences, Darmstadt, Germany), orPeptech (Burlington, Mass., USA). Those that are not commerciallyavailable are optionally synthesized as provided herein or usingstandard methods known to those of ordinary skill in the art. Fororganic synthesis techniques, see, e.g., Organic Chemistry by Fessendonand Fessendon, (1982, Second Edition, Willard Grant Press, BostonMass.); Advanced Organic Chemistry by March (Third Edition, 1985, Wileyand Sons, New York); and Advanced Organic Chemistry by Carey andSundberg (Third Edition, Parts A and B, 1990, Plenum Press, New York).See, also, U.S. Patent Application Publications 2003/0082575 and2003/0108885, which are incorporated by reference herein. In addition tounnatural amino acids that contain novel side chains, unnatural aminoacids that may be suitable for use in the present invention alsooptionally comprise modified backbone structures, including but notlimited to, as illustrated by the structures of Formula II and III:

wherein Z typically comprises OH, NH₂, SH, NH—R′, or S—R′; X and Y,which can be the same or different, typically comprise S or O, and R andR′, which are optionally the same or different, are typically selectedfrom the same list of constituents for the R group described above forthe unnatural amino acids having Formula I as well as hydrogen. Forexample, unnatural amino acids of the invention optionally comprisesubstitutions in the amino or carboxyl group as illustrated by FormulasII and III. Unnatural amino acids of this type include, but are notlimited to, α-hydroxy acids, α-thioacids, α-aminothiocarboxylates,including but not limited to, with side chains corresponding to thecommon twenty natural amino acids or unnatural side chains. In addition,substitutions at the α-carbon optionally include but are not limited to,L, D, or α-α-disubstituted amino acids such as D-glutamate, D-alanine,D-methyl-O-tyrosine, aminobutyric acid, and the like. Other structuralalternatives include cyclic amino acids, such as proline analogues aswell as 3, 4, 6, 7, 8, and 9 membered ring proline analogues, β and γamino acids such as substituted β-alanine and γ-amino butyric acid.

Many unnatural amino acids are based on natural amino acids, such astyrosine, glutamine, phenylalanine, and the like, and are suitable foruse in the present invention. Tyrosine analogs include, but are notlimited to, para-substituted tyrosines, ortho-substituted tyrosines, andmeta substituted tyrosines, where the substituted tyrosine comprises,including but not limited to, a keto group (including but not limitedto, an acetyl group), a benzoyl group, an amino group, a hydrazine, anhydroxyamine, a thiol group, a carboxy group, an isopropyl group, amethyl group, a C₆-C₂₀ straight chain or branched hydrocarbon, asaturated or unsaturated hydrocarbon, an O-methyl group, a polyethergroup, a nitro group, an alkynyl group or the like. In addition,multiply substituted aryl rings are also contemplated. Glutamine analogsthat may be suitable for use in the present invention include, but arenot limited to, α-hydroxy derivatives, γ-substituted derivatives, cyclicderivatives, and amide substituted glutamine derivatives. Examplephenylalanine analogs that may be suitable for use in the presentinvention include, but are not limited to, para-substitutedphenylalanines, ortho-substituted phenyalanines, and meta-substitutedphenylalanines, where the substituent comprises, including but notlimited to, a hydroxy group, a methoxy group, a methyl group, an allylgroup, an aldehyde, an azido, an iodo, a bromo, a keto group (includingbut not limited to, an acetyl group), a benzoyl, an alkynyl group, orthe like. Specific examples of unnatural amino acids that may besuitable for use in the present invention include, but are not limitedto, a p-acetyl-L-phenylalanine, an O-methyl-L-tyrosine, anL-3-(2-naphthyl)alanine, a 3-methyl-phenylalanine, anO-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine, atri-O-acetyl-GlcNAcβ-serine, an L-Dopa, a fluorinated phenylalanine, anisopropyl-L-phenylalanine, a p-azido-L-phenylalanine, ap-acyl-L-phenylalanine, a p-benzoyl-L-phenylalanine, an L-phosphoserine,a phosphonoserine, a phosphonotyrosine, a p-iodo-phenylalanine, ap-bromophenylalanine, a p-amino-L-phenylalanine, anisopropyl-L-phenylalanine, and a p-propargyloxy-phenylalanine, and thelike. Examples of structures of a variety of unnatural amino acids thatmay be suitable for use in the present invention are provided in, forexample, WO 2002/085923 entitled “In vivo incorporation of unnaturalamino acids.” See also Kiick et al., (2002) Incorporation of azides intorecombinant proteins for chemoselective modification by the Staudingerligation, PNAS 99:19-24, which is incorporated by reference herein, foradditional methionine analogs.

In one embodiment, compositions of a hGH polypeptide that include anunnatural amino acid (such as p-(propargyloxy)-phenyalanine) areprovided. Various compositions comprising p-(propargyloxy)-phenyalanineand, including but not limited to, proteins and/or cells, are alsoprovided. In one aspect, a composition that includes thep-(propargyloxy)-phenyalanine unnatural amino acid, further includes anorthogonal tRNA. The unnatural amino acid can be bonded (including butnot limited to, covalently) to the orthogonal tRNA, including but notlimited to, covalently bonded to the orthogonal tRNA though anamino-acyl bond, covalently bonded to a 3'OH or a 2'OH of a terminalribose sugar of the orthogonal tRNA, etc.

The chemical moieties via unnatural amino acids that can be incorporatedinto proteins offer a variety of advantages and manipulations of theprotein: For example, the unique reactivity of a keto functional groupallows selective modification of proteins with any of a number ofhydrazine- or hydroxylamine-containing reagents in vitro and in vivo. Aheavy atom unnatural amino acid, for example, can be useful for phasingX-ray structure data. The site-specific introduction of heavy atomsusing unnatural amino acids also provides selectivity and flexibility inchoosing positions for heavy atoms. Photoreactive unnatural amino acids(including but not limited to, amino acids with benzophenone andarylazides (including but not limited to, phenylazide) side chains), forexample, allow for efficient in vivo and in vitro photocrosslinking ofprotein. Examples of photoreactive unnatural amino acids include, butare not limited to, p-azido-phenylalanine and p-benzoyl-phenylalanine.The protein with the photoreactive unnatural amino acids can then becrosslinked at will by excitation of the photoreactive group-providingtemporal control. In one example, the methyl group of an unnatural aminocan be substituted with an isotopically labeled, including but notlimited to, methyl group, as a probe of local structure and dynamics,including but not limited to, with the use of nuclear magnetic resonanceand vibrational spectroscopy. Alkynyl or azido functional groups, forexample, allow the selective modification of proteins with moleculesthrough a [3+2]cycloaddition reaction.

A non-natural amino acid incorporated into a polypeptide at the aminoterminus can be composed of an R group that is any substituent otherthan one used in the twenty natural amino acids and a 2^(nd) reactivegroup different from the NH₂ group normally present in α-amino acids(see Formula I). A similar non-natural amino acid can be incorporated atthe carboxyl terminus with a 2^(nd) reactive group different from theCOOH group normally present in α-amino acids (see Formula I).

The unnatural amino acids of the invention may be selected or designedto provide additional characteristics unavailable in the twenty naturalamino acids. For example, unnatural amino acid may be optionallydesigned or selected to modify the biological properties of a protein,e.g., into which they are incorporated. For example, the followingproperties may be optionally modified by inclusion of an unnatural aminoacid into a protein: toxicity, biodistribution, solubility, stability,e.g., thermal, hydrolytic, oxidative, resistance to enzymaticdegradation, and the like, facility of purification and processing,structural properties, spectroscopic properties, chemical and/orphotochemical properties, catalytic activity, redox potential,half-life, ability to react with other molecules, e.g., covalently ornoncovalently, and the like. U.S. patent application Ser. No.11/046,432, which is incorporated by reference herein, discusses anumber of different non-naturally encoded amino acids.

Chemical Synthesis of Unnatural Amino Acids

Many of the unnatural amino acids suitable for use in the presentinvention are commercially available, e.g., from Sigma (USA) or Aldrich(Milwaukee, Wis., USA). Those that are not commercially available areoptionally synthesized as provided herein or as provided in variouspublications or using standard methods known to those of ordinary skillin the art. For organic synthesis techniques, see, e.g., OrganicChemistry by Fessendon and Fessendon, (1982, Second Edition, WillardGrant Press, Boston Mass.); Advanced Organic Chemistry by March (ThirdEdition, 1985, Wiley and Sons, New York); and Advanced Organic Chemistryby Carey and Sundberg (Third Edition, Parts A and B, 1990, Plenum Press,New York). Additional publications describing the synthesis of unnaturalamino acids include, e.g., WO 2002/085923 entitled “In vivoincorporation of Unnatural Amino Acids;” Matsoukas et al., (1995) J.Med. Chem., 38, 4660-4669; King, F. E. & Kidd, D. A. A. (1949) A NewSynthesis of Glutamine and of γ-Dipeptides of Glutamic Acid fromPhthylated Intermediates. J. Chem. Soc., 3315-3319; Friedman, O. M. &Chatterrji, R. (1959) Synthesis of Derivatives of Glutamine as ModelSubstrates for Anti-Tumor Agents. J. Am. Chem. Soc. 81, 3750-3752;Craig, J. C. et al. (1988) Absolute Configuration of the Enantiomers of7-Chloro-4 [[4-(diethylamino)-J-methylbutyl]amino]quinoline(Chloroquine). J. Org. Chem. 53, 1167-1170; Azoulay, M., Vilmont, M. &Frappier, F. (1991) Glutamine analogues as Potential Antimalarials, Eur.J. Med. Chem. 26, 201-5; Koskinen, A. M. P. & Rapoport, H. (1989)Synthesis of 4-Substituted Prolines as Conformationally ConstrainedAmino Acid Analogues. J. Org. Chem. 54, 1859-1866; Christie, B. D. &Rapoport, H. (1985) Synthesis of Optically Pure Pipecolates fromL-Asparagine. Application to the Total Synthesis of (+)-Apovincaminethrough Amino Acid Decarbonylation and Iminium Ion Cyclization. J. Org.Chem. 50:1239-1246; Barton et al., (1987) Synthesis of Novelalpha-Amino-Acids and Derivatives Using Radical Chemistry: Synthesis ofL- and D-alpha-Amino-Adipic Acids, L-alpha-aminopimelic Acid andAppropriate Unsaturated Derivatives. Tetrahedron 43:4297-4308; and,Subasinghe et al., (1992) Quisqualic acid analogues: synthesis ofbeta-heterocyclic 2-aminopropanoic acid derivatives and their activityat a novel quisqualate-sensitized site. J. Med. Chem. 35:4602-7. Seealso, U.S. Patent Publication No. US 2004/0198637 entitled “ProteinArrays,” which is incorporated by reference.

For example, the synthesis of p-acetyl-(+/−)-phenylalanine andm-acetyl-(+/−)-phenylalanine is described in Zhang, Z., et al.,Biochemistry 42: 6735-6746 (2003), which is incorporated by referenceherein. Other carbonyl-containing amino acids can be similarly preparedby one of ordinary skill in the art. The carbonyl functionality can bereacted selectively with a hydrazine-, hydrazide-, hydroxylamine-, orsemicarbazide-containing reagent under mild conditions in aqueoussolution to form the corresponding hydrazone, oxime, or semicarbazonelinkages, respectively, that are stable under physiological conditions.See, e.g., Jencks, W. P., J. Am. Chem. Soc. 81, 475-481 (1959); Shao, J.and Tam, J. P., J. Am. Chem. Soc. 117:3893-3899 (1995). Moreover, theunique reactivity of the carbonyl group allows for selectivemodification in the presence of the other amino acid side chains. See,e.g., Cornish, V. W., et al., J. Am. Chem. Soc. 118:8150-8151 (1996);Geoghegan, K. F. & Stroh, J. G., Bioconjug. Chem. 3:138-146 (1992);Mahal, L. K., et al., Science 276:1125-1128 (1997).

Biosynthesis of Unnatural Amino Acids

Many biosynthetic pathways already exist in cells for the production ofamino acids and other compounds. While a biosynthetic method for aparticular unnatural amino acid may not exist in nature, including butnot limited to, in a cell, the invention provides such methods. Forexample, biosynthetic pathways for unnatural amino acids are optionallygenerated in host cell by adding new enzymes or modifying existing hostcell pathways. Additional new enzymes are optionally naturally occurringenzymes or artificially evolved enzymes. For example, the biosynthesisof p-aminophenylalanine (as presented in an example in WO 2002/085923entitled “In vivo incorporation of unnatural amino acids”) relies on theaddition of a combination of known enzymes from other organisms. Thegenes for these enzymes can be introduced into a eukaryotic cell bytransforming the cell with a plasmid comprising the genes. The genes,when expressed in the cell, provide an enzymatic pathway to synthesizethe desired compound. Examples of the types of enzymes that areoptionally added are provided in the examples below. Additional enzymessequences are found, for example, in Genbank. Artificially evolvedenzymes are also optionally added into a cell in the same manner. Inthis manner, the cellular machinery and resources of a cell aremanipulated to produce unnatural amino acids.

A variety of methods are available for producing novel enzymes for usein biosynthetic pathways or for evolution of existing pathways. Forexample, recursive recombination, including but not limited to, asdeveloped by Maxygen, Inc. (available on the World Wide Web atmaxygen.com), is optionally used to develop novel enzymes and pathways.See, e.g., Stemmer (1994), Rapid evolution of a protein in vitro by DNAshuffling, Nature 370(4):389-391; and, Stemmer, (1994), DNA shuffling byrandom fragmentation and reassembly: In vitro recombination formolecular evolution, Proc. Natl. Acad. Sci. USA., 91:10747-10751.Similarly DESIGNPATH©, developed by Genencor (available on the WorldWide Web at genencor.com) is optionally used for metabolic pathwayengineering, including but not limited to, to engineer a pathway tocreate O-methyl-L-tyrosine in a cell. This technology reconstructsexisting pathways in host organisms using a combination of new genes,including but not limited to, those identified through functionalgenomics, and molecular evolution and design. Diversa Corporation(available on the World Wide Web at diversa.com) also providestechnology for rapidly screening libraries of genes and gene pathways,including but not limited to, to create new pathways.

Typically, the unnatural amino acid produced with an engineeredbiosynthetic pathway of the invention is produced in a concentrationsufficient for efficient protein biosynthesis, including but not limitedto, a natural cellular amount, but not to, such a degree as to affectthe concentration of the other amino acids or exhaust cellularresources. Typical concentrations produced in vivo in this manner areabout 10 mM to about 0.05 mM. Once a cell is transformed with a plasmidcomprising the genes used to produce enzymes desired for a specificpathway and an unnatural amino acid is generated, in vivo selections areoptionally used to further optimize the production of the unnaturalamino acid for both ribosomal protein synthesis and cell growth.

Cellular Uptake Of Unnatural Amino Acids

Unnatural amino acid uptake by a cell is one issue that is typicallyconsidered when designing and selecting unnatural amino acids, includingbut not limited to, for incorporation into a protein. For example, thehigh charge density of α-amino acids suggests that these compounds areunlikely to be cell permeable. Natural amino acids are taken up into theeukaryotic cell via a collection of protein-based transport systems. Arapid screen can be done which assesses which unnatural amino acids, ifany, are taken up by cells. See, e.g., the toxicity assays in, e.g.,U.S. Patent Publication No. US 2004/0198637 entitled “Protein. Arrays,”which is incorporated by reference herein; and Liu, D. R. & Schultz, P.G. (1999) Progress toward the evolution of an organism with an expandedgenetic code. PNAS United States 96:4780-4785. Although uptake is easilyanalyzed with various assays, an alternative to designing unnaturalamino acids that are amenable to cellular uptake pathways is to providebiosynthetic pathways to create amino acids in vivo.

V. Polypeptides with Unnatural Amino Acids

The incorporation of an unnatural amino acid can be done for a varietyof purposes, including but not limited to, tailoring changes in proteinstructure and/or function, changing size, acidity, nucleophilicity,hydrogen bonding, hydrophobicity, accessibility of protease targetsites, targeting to a moiety (including but not limited to, for aprotein array), adding a biologically active molecule, attaching apolymer, attaching a radionuclide, modulating serum half-life,modulating tissue penetration (e.g. tumors), modulating activetransport, modulating tissue, cell or organ specificity or distribution,modulating immunogenicity, modulating protease resistance, etc. Proteinsthat include an unnatural amino acid can have enhanced or even entirelynew catalytic or biophysical properties. For example, the followingproperties are optionally modified by inclusion of an unnatural aminoacid into a protein: toxicity, biodistribution, structural properties,spectroscopic properties, chemical and/or photochemical properties,catalytic ability, half-life (including but not limited to, serumhalf-life), ability to react with other molecules, including but notlimited to, covalently or noncovalently, and the like. The compositionsincluding proteins that include at least one unnatural amino acid areuseful for, including but not limited to, novel therapeutics,diagnostics, catalytic enzymes, industrial enzymes, binding proteins(including but not limited to, antibodies), and including but notlimited to, the study of protein structure and function. See, e.g.,Dougherty, (2000) Unnatural Amino Acids as Probes of Protein Structureand Function, Current Opinion in Chemical Biology, 4:645-652.

In one aspect of the invention, a composition includes at least oneprotein with at least one, including but not limited to, at least two,at least three, at least four, at least five, at least six, at leastseven, at least eight, at least nine, or at least ten or more unnaturalamino acids. The unnatural amino acids can be the same or different,including but not limited to, there can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or10 or more different sites in the protein that comprise 1, 2, 3, 4, 5,6, 7, 8, 9, or 10 or more different unnatural amino acids. In anotheraspect, a composition includes a protein with at least one, but fewerthan all, of a particular amino acid present in the protein issubstituted with the unnatural amino acid. For a given protein with morethan one unnatural amino acids, the unnatural amino acids can beidentical or different (including but not limited to, the protein caninclude two or more different types of unnatural amino acids, or caninclude two of the same unnatural amino acid). For a given protein withmore than two unnatural amino acids, the unnatural amino acids can bethe same, different or a combination of a multiple unnatural amino acidof the same kind with at least one different unnatural amino acid.

Proteins or polypeptides of interest with at least one unnatural aminoacid are a feature of the invention. The invention also includespolypeptides or proteins with at least one unnatural amino acid producedusing the compositions and methods of the invention. An excipient(including but not limited to, a pharmaceutically acceptable excipient)can also be present with the protein.

By producing proteins or polypeptides of interest with at least oneunnatural amino acid in eukaryotic cells, proteins or polypeptides willtypically include eukaryotic post-translational modifications. Incertain embodiments, a protein includes at least one unnatural aminoacid and at least one post-translational modification that is made invivo by a eukaryotic cell, where the post-translational modification isnot made by a prokaryotic cell. For example, the post-translationmodification includes, including but not limited to, acetylation,acylation, lipid-modification, palmitoylation, palmitate addition,phosphorylation, glycolipid-linkage modification, glycosylation, and thelike. In one aspect, the post-translational modification includesattachment of an oligosaccharide (including but not limited to,(GlcNAc-Man)₂-Man-GlcNAc-GlcNAc)) to an asparagine by aGlcNAc-asparagine linkage. See Table 1 which lists some examples ofN-linked oligosaccharides of eukaryotic proteins (additional residuescan also be present, which are not shown). In another aspect, thepost-translational modification includes attachment of anoligosaccharide (including but not limited to, Gal-GalNAc, Gal-GlcNAc,etc.) to a serine or threonine by a GalNAc-serine or GalNAc-threoninelinkage, or a GlcNAc-serine or a GlcNAc-threonine linkage.

TABLE 1 Examples of oligosaccharides through GLCNAC-linkage Type BaseStructure I. High-mannose

II. Hybrid

III. Complex

IV. Xylose

In yet another aspect, the post-translation modification includesproteolytic processing of precursors, assembly into a multisubunitprotein or macromolecular assembly, translation to another site in thecell (including but not limited to, to organelles, such as theendoplasmic reticulum, the Golgi apparatus, the nucleus, lysosomes,peroxisomes, mitochondria, chloroplasts, vacuoles, etc., or through thesecretory pathway). In certain embodiments, the protein comprises asecretion or localization sequence, an epitope tag, a FLAG tag, apolyhistidine tag, a GST fusion, or the like. U.S. Pat. Nos. 4,963,495and 6,436,674, which are incorporated herein by reference, detailconstructs designed to improve secretion of hGH polypeptides.

One advantage of an unnatural amino acid is that it presents additionalchemical moieties that can be used to add additional molecules. Thesemodifications can be made in vivo in a eukaryotic or non-eukaryoticcell, or in vitro. Thus, in certain embodiments, the post-translationalmodification is through the unnatural amino acid. For example, thepost-translational modification can be through anucleophilic-electrophilic reaction. Most reactions currently used forthe selective modification of proteins involve covalent bond formationbetween nucleophilic and electrophilic reaction partners, including butnot limited to the reaction of α-haloketones with histidine or cysteineside chains. Selectivity in these cases is determined by the number andaccessibility of the nucleophilic residues in the protein. In proteinsof the invention, other more selective reactions can be used such as thereaction of an unnatural keto-amino acid with hydrazides or aminooxycompounds, in vitro and in vivo. See, e.g., Cornish, et al., (1996) J.Am. Chem. Soc., 118:8150-8151; Mahal, et al., (1997) Science,276:1125-1128; Wang, et al., (2001) Science 292:498-500; Chin, et al.,(2002) J. Am. Chem. Soc. 124:9026-9027; Chin, et al., (2002) Proc. Natl.Acad. Sci., 99:11020-11024; Wang, et al., (2003) Proc. Natl. Acad. Sci.,100:56-61; Zhang, et al., (2003) Biochemistry, 42:6735-6746; and, Chin,et al., (2003) Science, 301:964-7, all of which are incorporated byreference herein. This allows the selective labeling of virtually anyprotein with a host of reagents including fluorophores, crosslinkingagents, saccharide derivatives and cytotoxic molecules. See also, U.S.Pat. No. 6,927,042 entitled “Glycoprotein Synthesis,” which isincorporated by reference herein. Post-translational modifications,including but not limited to, through an azido amino acid, can also madethrough the Staudinger ligation (including but not limited to, withtriarylphosphine reagents). See, e.g., Kiick et al., (2002)Incorporation of azides into recombinant proteins for chemoselectivemodification by the Staudinger ligation, PNAS 99:19-24.

This invention provides another highly efficient method for theselective modification of proteins, which involves the geneticincorporation of unnatural amino acids. A molecule that can be added toa protein include, but are not limited to, dyes, fluorophores,crosslinking agents, saccharide derivatives, polymers (including but notlimited to, derivatives of polyethylene glycol), photocrosslinkers,cytotoxic compounds, affinity labels, derivatives of biotin, resins,beads, a second protein or polypeptide (or more), polynucleotide(s)(including but not limited to, DNA, RNA, etc.), metal chelators,cofactors, fatty acids, carbohydrates, and the like. In one embodiment,the method further includes incorporating into the protein the unnaturalamino acid, where the unnatural amino acid comprises a first reactivegroup; and contacting the protein with a molecule (including but notlimited to, a label, a dye, a polymer, a water-soluble polymer, aderivative of polyethylene glycol, a photocrosslinker, a radionuclide, acytotoxic compound, a drug, an affinity label, a photoaffinity label, areactive compound, a resin, a second protein or polypeptide orpolypeptide analog, an antibody or antibody fragment, a metal chelator,a cofactor, a fatty acid, a carbohydrate, a polynucleotide, a DNA, aRNA, an antisense polynucleotide, a water-soluble dendrimer, acyclodextrin, an inhibitory ribonucleic acid, a saccharide, abiomaterial, a nanoparticle, a spin label, a fluorophore, ametal-containing moiety, a radioactive moiety, a novel functional group,a group that covalently or noncovalently interacts with other molecules,a photocaged moiety, an actinic radiation excitable moiety, aphotoisomerizable moiety, biotin, a derivative of biotin, a biotinanalogue, a moiety incorporating a heavy atom, a chemically cleavablegroup, a photocleavable group, an elongated side chain, a carbon-linkedsugar, a redox-active agent, an amino thioacid, a toxic moiety, anisotopically labeled moiety, a biophysical probe, a phosphorescentgroup, a chemiluminescent group, an electron dense group, a magneticgroup, an intercalating group, a chromophore, an energy transfer agent,a biologically active agent, a detectable label, a small molecule, aquantum dot, a nanotransmitter, a radionucleotide, a radiotransmitter, aneutron-capture agent, or any combination of the above, or any otherdesirable compound or substance) that comprises a second reactive group.

VI. In Vivo Generation of hGH Polypeptides ComprisingNon-Genetically-Encoded Amino Acids

The hGH polypeptides of the invention can be generated in vivo usingmodified tRNA and tRNA synthetases to add to or substitute amino acidsthat are not encoded in naturally-occurring systems.

Methods for generating tRNAs and tRNA synthetases which use amino acidsthat are not encoded in naturally-occurring systems are described in,e.g., U.S. Patent Application Publications 2003/0082575 (Ser. No.10/126,927) and 2003/0108885 (Ser. No. 10/126,931) which areincorporated by reference herein. These methods involve generating atranslational machinery that functions independently of the synthetasesand tRNAs endogenous to the translation system (and are thereforesometimes referred to as “orthogonal”). Typically, the translationsystem comprises an orthogonal tRNA (O-tRNA) and an orthogonal aminoacyltRNA synthetase (O-RS). Typically, the O-RS preferentially aminoacylatesthe O-tRNA with at least one non-naturally occurring amino acid in thetranslation system and the O-tRNA recognizes at least one selector codonthat is not recognized by other tRNAs in the system. The translationsystem thus inserts the non-naturally-encoded amino acid into a proteinproduced in the system, in response to an encoded selector codon,thereby “substituting” an amino acid into a position in the encodedpolypeptide.

A wide variety of orthogonal tRNAs and aminoacyl tRNA synthetases havebeen described in the art for inserting particular synthetic amino acidsinto polypeptides, and are generally suitable for use in the presentinvention. For example, keto-specific O-tRNA/aminoacyl-tRNA synthetasesare described in Wang, L., et al., Proc. Natl. Acad. Sci. USA 100:56-61(2003) and Zhang, Z. et al., Biochem. 42(22):6735-6746 (2003). ExemplaryO-RS, or portions thereof, are encoded by polynucleotide sequences andinclude amino acid sequences disclosed in U.S. Patent ApplicationPublications 2003/0082575 and 2003/0108885, each incorporated herein byreference. Corresponding O-tRNA molecules for use with the O-RSs arealso described in U.S. Patent Application Publications 2003/0082575(Ser. No. 10/126,927) and 2003/0108885 (Ser. No. 10/126,931) which areincorporated by reference herein.

An example of an azide-specific O-tRNA/aminoacyl-tRNA synthetase systemis described in Chin, J. W., et al., J. Am. Chem. Soc. 124:9026-9027(2002). Exemplary O-RS sequences forp-azido-L-Phe include, but are notlimited to, nucleotide sequences SEQ ID NOs: 14-16 and 29-32 and aminoacid sequences SEQ ID NOs: 46-48 and 61-64 as disclosed in U.S. PatentApplication Publication 2003/0108885 (Ser. No. 10/126,931) which isincorporated by reference herein. Exemplary O-tRNA sequences suitablefor use in the present invention include, but are not limited to,nucleotide sequences SEQ ID NOs: 1-3 as disclosed in U.S. PatentApplication Publication 2003/0108885 (Ser. No. 10/126,931) which isincorporated by reference herein. Other examples ofO-tRNA/aminoacyl-tRNA synthetase pairs specific to particularnon-naturally encoded amino acids are described in U.S. PatentApplication Publication 2003/0082575 (Ser. No. 10/126,927) which isincorporated by reference herein. O-RS and O-tRNA that incorporate bothketo- and azide-containing amino acids in S. cerevisiae are described inChin, J. W., et al., Science 301:964-967 (2003).

Several other orthogonal pairs have been reported. Glutaminyl (see,e.g., Liu, D. R., and Schultz, P. G. (1999) Proc. Natl. Acad. Sci.U.S.A. 96:4780-4785), aspartyl (see, e.g., Pastmak, M., et al., (2000)Helv. Chim. Acta 83:2277-2286), and tyrosyl (see, e.g., Ohno, S., etal., (1998) J. Biochem. (Tokyo, Jpn.) 124:1065-1068; and, Kowal, A. K.,et al., (2001) Proc. Natl. Acad. Sci. U.S.A. 98:2268-2273) systemsderived from S. cerevisiae tRNA's and synthetases have been describedfor the potential incorporation of unnatural amino acids in E. coli.Systems derived from the E. coli glutaminyl (see, e.g., Kowal, A. K., etal., (2001) Proc. Natl. Acad. Sci. U.S.A. 98:2268-2273) and tyrosyl(see, e.g., Edwards, H., and Schimmel, P. (1990) Mol. Cell. Biol.10:1633-1641) synthetases have been described for use in S. cerevisiae.The E. coli tyrosyl system has been used for the incorporation of3-iodo-L-tyrosine in vivo, in mammalian cells. See, Sakamoto, K., etal., (2002) Nucleic Acids Res. 30:4692-4699.

Use of O-tRNA/aminoacyl-tRNA synthetases involves selection of aspecific codon which encodes the non-naturally encoded amino acid. Whileany codon can be used, it is generally desirable to select a codon thatis rarely or never used in the cell in which the O-tRNA/aminoacyl-tRNAsynthetase is expressed. For example, exemplary codons include nonsensecodon such as stop codons (amber, ochre, and opal), four or more basecodons and other natural three-base codons that are rarely or unused.

Specific selector codon(s) can be introduced into appropriate positionsin the hGH polynucleotide coding sequence using mutagenesis methodsknown in the art (including but not limited to, site-specificmutagenesis, cassette mutagenesis, restriction selection mutagenesis,etc.).

Methods for generating components of the protein biosynthetic machinery,such as O-RSs, O-tRNAs, and orthogonal O-tRNA/O-RS pairs that can beused to incorporate a non-naturally encoded amino acid are described inWang, L., et al., Science 292: 498-500 (2001); Chin, J. W., et al., J.Am. Chem. Soc. 124:9026-9027 (2002); Zhang, Z. et al., Biochemistry 42:6735-6746 (2003): Methods and compositions for the in vivo incorporationof non-naturally encoded amino acids are described in U.S. PatentApplication Publication 2003/0082575 (Ser. No. 10/126,927) which isincorporated by reference herein. Methods for selecting an orthogonaltRNA-tRNA synthetase pair for use in in vivo translation system of anorganism are also described in U.S. Patent Application Publications2003/0082575 (Ser. No. 10/126,927) and 2003/0108885 (Ser. No.10/126,931) which are incorporated by reference herein. PCT PublicationNo. WO 04/035743 entitled “Site Specific Incorporation of Keto AminoAcids into Proteins,” which is incorporated by reference herein in itsentirety, describes orthogonal RS and tRNA pairs for the incorporationof keto amino acids. PCT Publication No. WO 04/094593 entitled“Expanding the Eukaryotic Genetic Code,” which is incorporated byreference herein in its entirety, describes orthogonal RS and tRNA pairsfor the incorporation of non-naturally encoded amino acids in eukaryotichost cells.

Methods for producing at least one recombinant orthogonal aminoacyl-tRNAsynthetase (O-RS) comprise: (a) generating a library of (optionallymutant) RSs derived from at least one aminoacyl-tRNA synthetase (RS)from a first organism, including but not limited to, a prokaryoticorganism, such as Methanococcus jannaschii, Methanobacteriumthermoautotrophicum, Halobacterium, Escherichia coli, A. fulgidus, P.furiosus, P. horikoshii, A. pernix, T. thermophilus, or the like, or aeukaryotic organism; (b) selecting (and/or screening) the library of RSs(optionally mutant RSs) for members that aminoacylate an orthogonal tRNA(O-tRNA) in the presence of a non-naturally encoded amino acid and anatural amino acid, thereby providing a pool of active (optionallymutant) RSs; and/or, (c) selecting (optionally through negativeselection) the pool for active RSs (including but not limited to, mutantRSs) that preferentially aminoacylate the O-tRNA in the absence of thenon-naturally encoded amino acid, thereby providing the at least onerecombinant O-RS; wherein the at least one recombinant O-RSpreferentially aminoacylates the O-tRNA with the non-naturally encodedamino acid.

In one embodiment, the RS is an inactive RS. The inactive RS can begenerated by mutating an active RS. For example, the inactive RS can begenerated by mutating at least about 1, at least about 2, at least about3, at least about 4, at least about 5, at least about 6, or at leastabout 10 or more amino acids to different amino acids, including but notlimited to, alanine.

Libraries of mutant RSs can be generated using various techniques knownin the art, including but not limited to rational design based onprotein three dimensional RS structure, or mutagenesis of RS nucleotidesin a random or rational design technique. For example, the mutant RSscan be generated by site-specific mutations, random mutations, diversitygenerating recombination mutations, chimeric constructs, rational designand by other methods described herein or known in the art.

In one embodiment, selecting (and/or screening) the library of RSs(optionally mutant RSs) for members that are active, including but notlimited to, that aminoacylate an orthogonal tRNA (O-tRNA) in thepresence of a non-naturally encoded amino acid and a natural amino acid,includes: introducing a positive selection or screening marker,including but not limited to, an antibiotic resistance gene, or thelike, and the library of (optionally mutant) RSs into a plurality ofcells, wherein the positive selection and/or screening marker comprisesat least one selector codon, including but not limited to, an amber,ochre, or opal codon; growing the plurality of cells in the presence ofa selection agent; identifying cells that survive (or show a specificresponse) in the presence of the selection and/or screening agent bysuppressing the at least one selector codon in the positive selection orscreening marker, thereby providing a subset of positively selectedcells that contains the pool of active (optionally mutant) RSs.Optionally, the selection and/or screening agent concentration can bevaried.

In one aspect, the positive selection marker is a chloramphenicolacetyltransferase (CAT) gene and the selector codon is an amber stopcodon in the CAT gene. Optionally, the positive selection marker is aβ-lactamase gene and the selector codon is an amber stop codon in theβ-lactamase gene. In another aspect the positive screening markercomprises a fluorescent or luminescent screening marker or an affinitybased screening marker (including but not limited to, a cell surfacemarker).

In one embodiment, negatively selecting or screening the pool for activeRSs (optionally mutants) that preferentially aminoacylate the O-tRNA inthe absence of the non-naturally encoded amino acid includes:introducing a negative selection or screening marker with the pool ofactive (optionally mutant) RSs from the positive selection or screeninginto a plurality of cells of a second organism, wherein the negativeselection or screening marker comprises at least one selector codon(including but not limited to, an antibiotic resistance gene, includingbut not limited to, a chloramphenicol acetyltransferase (CAT) gene);and, identifying cells that survive or show a specific screeningresponse in a first medium supplemented with the non-naturally encodedamino acid and a screening or selection agent, but fail to survive or toshow the specific response in a second medium not supplemented with thenon-naturally encoded amino acid and the selection or screening agent,thereby providing surviving cells or screened cells with the at leastone recombinant O-RS. For example, a CAT identification protocoloptionally acts as a positive selection and/or a negative screening indetermination of appropriate O-RS recombinants. For instance, a pool ofclones is optionally replicated on growth plates containing CAT (whichcomprises at least one selector codon) either with or without one ormore non-naturally encoded amino acid. Colonies growing exclusively onthe plates containing non-naturally encoded amino acids are thusregarded as containing recombinant O-RS. In one aspect, theconcentration of the selection (and/or screening) agent is varied. Insome aspects the first and second organisms are different. Thus, thefirst and/or second organism optionally comprises: a prokaryote, aeukaryote, a mammal, an Escherichia coli, a fungi, a yeast, anarchaebacterium, a eubacterium, a plant, an insect, a protist, etc. Inother embodiments, the screening marker comprises a fluorescent orluminescent screening marker or an affinity based screening marker.

In another embodiment, screening or selecting (including but not limitedto, negatively selecting) the pool for active (optionally mutant) RSsincludes: isolating the pool of active mutant RSs from the positiveselection step (b); introducing a negative selection or screeningmarker, wherein the negative selection or screening marker comprises atleast one selector codon (including but not limited to, a toxic markergene, including but not limited to, a ribonuclease barnase gene,comprising at least one selector codon), and the pool of active(optionally mutant) RSs into a plurality of cells of a second organism;and identifying cells that survive or show a specific screening responsein a first medium not supplemented with the non-naturally encoded aminoacid, but fail to survive or show a specific screening response in asecond medium supplemented with the non-naturally encoded amino acid,thereby providing surviving or screened cells with the at least onerecombinant O-RS, wherein the at least one recombinant O-RS is specificfor the non-naturally encoded amino acid. In one aspect, the at leastone selector codon comprises about two or more selector codons. Suchembodiments optionally can include wherein the at least one selectorcodon comprises two or more selector codons, and wherein the first andsecond organism are different (including but not limited to, eachorganism is optionally, including but not limited to, a prokaryote, aeukaryote, a mammal, an Escherichia coli, a fungi, a yeast, anarchaebacteria, a eubacteria, a plant, an insect, a protist, etc.).Also, some aspects include wherein the negative selection markercomprises a ribonuclease barnase gene (which comprises at least oneselector codon). Other aspects include wherein the screening markeroptionally comprises a fluorescent or luminescent screening marker or anaffinity based screening marker. In the embodiments herein, thescreenings and/or selections optionally include variation of thescreening and/or selection stringency.

In one embodiment, the methods for producing at least one recombinantorthogonal aminoacyl-tRNA synthetase (O-RS) can further comprise: (d)isolating the at least one recombinant O-RS; (e) generating a second setof O-RS (optionally mutated) derived from the at least one recombinantO-RS; and, (f) repeating steps (b) and (c) until a mutated O-RS isobtained that comprises an ability to preferentially aminoacylate theO-tRNA. Optionally, steps (d)-(f) are repeated, including but notlimited to, at least about two times. In one aspect, the second set ofmutated O-RS derived from at least one recombinant O-RS can be generatedby mutagenesis, including but not limited to, random mutagenesis,site-specific mutagenesis, recombination or a combination thereof.

The stringency of the selection/screening steps, including but notlimited to, the positive selection/screening step (b), the negativeselection/screening step (c) or both the positive and negativeselection/screening steps (b) and (c), in the above-described methods,optionally includes varying the selection/screening stringency. Inanother embodiment, the positive selection/screening step (b), thenegative selection/screening step (c) or both the positive and negativeselection/screening steps (b) and (c) comprise using a reporter, whereinthe reporter is detected by fluorescence-activated cell sorting (FACS)or wherein the reporter is detected by luminescence. Optionally, thereporter is displayed on a cell surface, on a phage display or the likeand selected based upon affinity or catalytic activity involving thenon-naturally encoded amino acid or an analogue. In one embodiment, themutated synthetase is displayed on a cell surface, on a phage display orthe like.

Methods for producing a recombinant orthogonal tRNA (O-tRNA) include:(a) generating a library of mutant tRNAs derived from at least one tRNA,including but not limited to, a suppressor tRNA, from a first organism;(b) selecting (including but not limited to, negatively selecting) orscreening the library for (optionally mutant) tRNAs that areaminoacylated by an aminoacyl-tRNA synthetase (RS) from a secondorganism in the absence of a RS from the first organism, therebyproviding a pool of tRNAs (optionally mutant); and, (c) selecting orscreening the pool of tRNAs (optionally mutant) for members that areaminoacylated by an introduced orthogonal RS (O-RS), thereby providingat least one recombinant O-tRNA; wherein the at least one recombinantO-tRNA recognizes a selector codon and is not efficiency recognized bythe RS from the second organism and is preferentially aminoacylated bythe O—RS. In some embodiments the at least one tRNA is a suppressor tRNAand/or comprises a unique three base codon of natural and/or unnaturalbases, or is a nonsense codon, a rare codon, an unnatural codon, a codoncomprising at least 4 bases, an amber codon, an ochre codon, or an opalstop codon. In one embodiment, the recombinant O-tRNA possesses animprovement of orthogonality. It will be appreciated that in someembodiments, O-tRNA is optionally imported into a first organism from asecond organism without the need for modification. In variousembodiments, the first and second organisms are either the same ordifferent and are optionally chosen from, including but not limited to,prokaryotes (including but not limited to, Methanococcus jannaschii,Methanobacterium thermoautotrophicum, Escherichia coli, Halobacterium,etc.), eukaryotes, mammals, fungi, yeasts, archaebacteria, eubacteria,plants, insects, protists, etc. Additionally, the recombinant tRNA isoptionally aminoacylated by a non-naturally encoded amino acid, whereinthe non-naturally encoded amino acid is biosynthesized in vivo eithernaturally or through genetic manipulation. The non-naturally encodedamino acid is optionally added to a growth medium for at least the firstor second organism.

In one aspect, selecting (including but not limited to, negativelyselecting) or screening the library for (optionally mutant) tRNAs thatare aminoacylated by an aminoacyl-tRNA synthetase (step (b)) includes:introducing a toxic marker gene, wherein the toxic marker gene comprisesat least one of the selector codons (or a gene that leads to theproduction of a toxic or static agent or a gene essential to theorganism wherein such marker gene comprises at least one selector codon)and the library of (optionally mutant) tRNAs into a plurality of cellsfrom the second organism; and, selecting surviving cells, wherein thesurviving cells contain the pool of (optionally mutant) tRNAs comprisingat least one orthogonal tRNA or nonfunctional tRNA. For example,surviving cells can be selected by using a comparison ratio cell densityassay.

In another aspect, the toxic marker gene can include two or moreselector codons. In another embodiment of the methods, the toxic markergene is a ribonuclease barnase gene, where the ribonuclease barnase genecomprises at least one amber codon. Optionally, the ribonuclease barnasegene can include two or more amber codons.

In one embodiment, selecting or screening the pool of (optionallymutant) tRNAs for members that are aminoacylated by an introducedorthogonal RS (O-RS) can include: introducing a positive selection orscreening marker gene, wherein the positive marker gene comprises a drugresistance gene (including but not limited to, β-lactamase gene,comprising at least one of the selector codons, such as at least oneamber stop codon) or a gene essential to the organism, or a gene thatleads to detoxification of a toxic agent, along with the O-RS, and thepool of (optionally mutant) tRNAs into a plurality of cells from thesecond organism; and, identifying surviving or screened cells grown inthe presence of a selection or screening agent, including but notlimited to, an antibiotic, thereby providing a pool of cells possessingthe at least one recombinant tRNA, where the at least one recombinanttRNA is aminoacylated by the O-RS and inserts an amino acid into atranslation product encoded by the positive marker gene, in response tothe at least one selector codons. In another embodiment, theconcentration of the selection and/or screening agent is varied.

Methods for generating specific O-tRNA/O-RS pairs are provided. Methodsinclude: (a) generating a library of mutant tRNAs derived from at leastone tRNA from a first organism; (b) negatively selecting or screeningthe library for (optionally mutant) tRNAs that are aminoacylated by anaminoacyl-tRNA synthetase (RS) from a second organism in the absence ofa RS from the first organism, thereby providing a pool of (optionallymutant) tRNAs; (c) selecting or screening the pool of (optionallymutant) tRNAs for members that are aminoacylated by an introducedorthogonal RS (O-RS), thereby providing at least one recombinant O-tRNA.The at least one recombinant O-tRNA recognizes a selector codon and isnot efficiency recognized by the RS from the second organism and ispreferentially aminoacylated by the O-RS. The method also includes (d)generating a library of (optionally mutant) RSs derived from at leastone aminoacyl-tRNA synthetase (RS) from a third organism; (e) selectingor screening the library of mutant RSs for members that preferentiallyaminoacylate the at least one recombinant O-tRNA in the presence of anon-naturally encoded amino acid and a natural amino, acid, therebyproviding a pool of active (optionally mutant) RSs; and, (f) negativelyselecting or screening the pool for active (optionally mutant) RSs thatpreferentially aminoacylate the at least one recombinant O-tRNA in theabsence of the non-naturally encoded amino acid, thereby providing theat least one specific O-tRNA/O-RS pair, wherein the at least onespecific O-tRNA/O-RS pair comprises at least one recombinant O-RS thatis specific for the non-naturally encoded amino acid and the at leastone recombinant O-tRNA. Specific O-tRNA/O-RS pairs produced by themethods are included. For example, the specific O-tRNA/O-RS pair caninclude, including but not limited to, a mutRNATyr-mutTyrRS pair, suchas a mutRNATyr-SS12TyrRS pair, a mutRNALeu-mutLeuRS pair, amutRNAThr-mutThrRS pair, a mutRNAGlu-mutGluRS pair, or the like.Additionally, such methods include wherein the first and third organismare the same (including but not limited to, Methanococcus jannaschii).

Methods for selecting an orthogonal tRNA-tRNA synthetase pair for use inan in vivo translation system of a second organism are also included inthe present invention. The methods include: introducing a marker gene, atRNA and an aminoacyl-tRNA synthetase (RS) isolated or derived from afirst organism into a first set of cells from the second organism;introducing the marker gene and the tRNA into a duplicate cell set froma second organism; and, selecting for surviving cells in the first setthat fail to survive in the duplicate cell set or screening for cellsshowing a specific screening response that fail to give such response inthe duplicate cell set, wherein the first set and the duplicate cell setare grown in the presence of a selection or screening agent, wherein thesurviving or screened cells comprise the orthogonal tRNA-tRNA synthetasepair for use in the in the in vivo translation system of the secondorganism. In one embodiment, comparing and selecting or screeningincludes an in vivo complementation assay. The concentration of theselection or screening agent can be varied.

The organisms of the present invention comprise a variety of organismand a variety of combinations. For example, the first and the secondorganisms of the methods of the present invention can be the same ordifferent. In one embodiment, the organisms are optionally a prokaryoticorganism, including but not limited to, Methanococcus jannaschii,Methanobacterium thermoautotrophicum, Halobacterium, Escherichia coli,A. fulgidus, P. furiosus, P. horikoshii, A. pernix, T thermophilus, orthe like. Alternatively, the organisms optionally comprise a eukaryoticorganism, including but not limited to, plants (including but notlimited to, complex plants such as monocots, or dicots), algae,protists, fungi (including but not limited to, yeast, etc), animals(including but not limited to, mammals, insects, arthropods, etc.), orthe like. In another embodiment, the second organism is a prokaryoticorganism, including but not limited to, Methanococcus jannaschii,Methanobacterium thermoautotrophicum, Halobacterium, Escherichia coli,A. fulgidus, Halobacterium, P. furiosus, P. horikoshii, A. pernix, T.thermophilus, or the like. Alternatively, the second organism can be aeukaryotic organism, including but not limited to, a yeast, a animalcell, a plant cell, a fungus, a mammalian cell, or the like. In variousembodiments the first and second organisms are different.

VII. Location of Non-Naturally-Occurring Amino Acids in hGH Polypeptides

The present invention contemplates incorporation of one or morenon-naturally-occurring amino acids into GH, e.g., hGH polypeptides. Oneor more non-naturally-occurring amino acids may be incorporated at aparticular position which does not disrupt activity of the polypeptide.This can be achieved by making “conservative” substitutions, includingbut not limited to, substituting hydrophobic amino acids withhydrophobic amino acids, bulky amino acids for bulky amino acids,hydrophilic amino acids for hydrophilic amino acids and/or inserting thenon-naturally-occurring amino acid in a location that is not requiredfor activity.

Regions of GH, e.g., hGH can be illustrated as follows, wherein theamino acid positions in hGH are indicated in the middle row (SEQ ID NO:2):

       Helix A             Helix B              HelixC                 Helix D [1-5] - [6-33] - [34-74] - [75-96] -[97-105] - [106-129] - [130-153] - [154-183] - [184-191]N-term         A-B loop             B-C loop                C-Dloop                C-term

A variety of biochemical and structural approaches can be employed toselect the desired sites for substitution with a non-naturally encodedamino acid within the GH, e.g., hGH polypeptide. It is readily apparentto those of ordinary skill in the art that any position of thepolypeptide chain is suitable for selection to incorporate anon-naturally encoded amino acid, and selection may be based on rationaldesign or by random selection for any or no particular desired purpose.Selection of desired sites may be for producing a GH, e.g., hGH moleculehaving any desired property or activity, including but not limited to,agonists, super-agonists, inverse agonists, antagonists, receptorbinding modulators, receptor activity modulators, dimer or multimerformation, no change to activity or property compared to the nativemolecule, or manipulating any physical or chemical property of thepolypeptide such as solubility, aggregation, or stability. For example,locations in the polypeptide required for biological activity of GH,e.g., hGH polypeptides can be identified using point mutation analysis,alanine scanning or homolog scanning methods known in the art. See,e.g., Cunningham, B. and Wells, J., Science, 244:1081-1085 (1989)(identifying 14 residues that are critical for GH, e.g., hGHbioactivity) and Cunningham, B., et al. Science 243: 1330-1336 (1989)(identifying antibody and receptor epitopes using homolog scanningmutagenesis). U.S. Pat. Nos. 5,580,723; 5,834,250; 6,013,478; 6,428,954;and 6,451,561, which are incorporated by reference herein, describemethods for the systematic analysis of the structure and function ofpolypeptides such as hGH by identifying active domains which influencethe activity of the polypeptide with a target substance. Residues otherthan those identified as critical to biological activity by alanine orhomolog scanning mutagenesis may be good candidates for substitutionwith a non-naturally encoded amino acid depending on the desiredactivity sought for the polypeptide. Alternatively, the sites identifiedas critical to biological activity may also be good candidates forsubstitution with a non-naturally encoded amino acid, again depending onthe desired activity sought for the polypeptide. Another alternativewould be to simply make serial substitutions in each position on thepolypeptide chain with a non-naturally encoded amino acid and observethe effect on the activities of the polypeptide. It is readily apparentto those of ordinary skill in the art that any means, technique, ormethod for selecting a position for substitution with a non-naturalamino acid into any polypeptide is suitable for use in the presentinvention.

The structure and activity of naturally-occurring mutants of hGHpolypeptides that contain deletions can also be examined to determineregions of the protein that are likely to be tolerant of substitutionwith a non-naturally encoded amino acid. See, e.g., Kostyo et al.,Biochem. Biophys. Acta, 925: 314 (1987); Lewis, U., et al., J. Biol.Chem., 253:2679-2687 (1978) for hGH. In a similar manner, proteasedigestion and monoclonal antibodies can be used to identify regions ofhGH that are responsible for binding the hGH receptor. See, e.g.,Cunningham, B., et al. Science 243: 1330-1336 (1989); Mills, J., et al.,Endocrinology, 107:391-399 (1980); Li, C., Mol. Cell. Biochem., 46:31-41(1982) (indicating that amino acids between residues 134-149 can bedeleted without a loss of activity). Once residues that are likely to beintolerant to substitution with non-naturally encoded amino acids havebeen eliminated, the impact of proposed substitutions at each of theremaining positions can be examined from the three-dimensional crystalstructure of the hGH and its binding proteins. See de Vos, A., et al.,Science, 255:306-312 (1992) for hGH; all crystal structures of hGH areavailable in the Protein Data Bank (including 3HHR, 1AXI, and 1HWG)(PDB, available on the World Wide Web at rcsb.org), a centralizeddatabase containing three-dimensional structural data of large moleculesof proteins and nucleic acids. Models may be made investigating thesecondary and tertiary structure of polypeptides, if three-dimensionalstructural data is not available. Thus, those of ordinary skill in theart can readily identify amino acid positions that can be substitutedwith non-naturally encoded amino acids.

In some embodiments, the GH, e.g., hGH polypeptides of the inventioncomprise one or more non-naturally occurring amino acids positioned in aregion of the protein that does not disrupt the helices or beta sheetsecondary structure of the polypeptide.

Exemplary residues of incorporation of a non-naturally encoded aminoacid may be those that are excluded from potential receptor bindingregions (including but not limited to, Site I and Site II), may be fullyor partially solvent exposed, have minimal or no hydrogen-bondinginteractions with nearby residues, may be minimally exposed to nearbyreactive residues, and may be in regions that are highly flexible(including but not limited to, C-D loop) or structurally rigid(including but not limited to, B helix) as predicted by thethree-dimensional, crystal structure, secondary, tertiary, or quaternarystructure of hGH, bound or unbound to its receptor.

In some embodiments, one or more non-naturally encoded amino acids areincorporated at any position in one or more of the following regionscorresponding to secondary structures in hGH as follows: positionscorresponding to 1-5 (N-terminus), 6-33 (A helix), 34-74 (region betweenA helix and B helix, the A-B loop), 75-96 (B helix), 97-105 (regionbetween B helix and C helix, the B-C loop), 106-129 (C helix), 130-153(region between C helix and D helix, the C-D loop), 154-183 (D helix),184-191 (C-terminus) from SEQ ID NO: 2. In other embodiments, GHpolypeptides, e.g., hGH polypeptides of the invention comprise at leastone non-naturally-occurring amino acid substituted for at least oneamino acid located in at least one region of GH, e.g., hGH selected fromthe group consisting regions corresponding to the N-terminus (1-5), theN-terminal end of the A-B loop (32-46); the B-C loop (97-105), the C-Dloop (132-149), and the C-terminus (184-191) of SEQ ID NO: 2. In someembodiments, one or more non-naturally encoded amino acids areincorporated at one or more of the following positions of GH, e.g., hGHcorresponding to: before position 1 (i.e. at the N-terminus), 1, 2, 3,4, 5, 8, 9, 11, 12, 15, 16, 19, 22, 29, 30, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 52, 55, 57, 59, 65, 66, 69,70, 71, 74, 88, 91, 92, 94, 95, 97, 98, 99, 100, 101, 102, 103, 104,105, 106, 107, 108, 109, 111, 112, 113, 115, 116, 119, 120, 122, 123,126, 127, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140,141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,155, 156, 158, 159, 161, 168, 172, 183, 184, 185, 186, 187, 188, 189,190, 191, 192 (i.e., at the carboxyl terminus of the protein) of SEQ IDNO: 2 or the corresponding amino acids of SEQ ID NO: 1 or 3.

Exemplary sites of incorporation of one or more non-naturally encodedamino acids include sites corresponding to 29, 30, 33, 34, 35, 37, 39,40, 49, 57, 59, 66, 69, 70, 71, 74, 88, 91, 92, 94, 95, 98, 99, 101,103, 107, 108, 111, 122, 126, 129, 130, 131, 133, 134, 135, 136, 137,139, 140, 141, 142, 143, 145, 147, 154, 155, 156, 159, 183, 186, and187, or any combination thereof from SEQ ID NO: 2 or the correspondingamino acids of SEQ ID NO: 1 or 3.

A subset of exemplary sites for incorporation of one or morenon-naturally encoded amino acid include sites corresponding to 29, 33,35, 37, 39, 49, 57, 69, 70, 71, 74, 88, 91, 92, 94, 95, 98, 99, 101,103, 107, 108, 111, 129, 130, 131, 133, 134, 135, 136, 137, 139, 140,141, 142, 143, 145, 147, 154, 155, 156, 186, and 187, or any combinationthereof from SEQ ID NO: 2 or the corresponding amino acids of SEQ ID NO:1 or 3. An examination of the crystal structure of GH, e.g., hGH and itsinteractions with the GH, e.g., hGH receptor indicates that the sidechains of these amino acid residues are fully or partially accessible tosolvent and the side chain of a non-naturally encoded amino acid maypoint away from the protein surface and out into the solvent.

Exemplary positions for incorporation of one or more non-naturallyencoded amino acids include sites corresponding to 35, 88, 91, 92, 94,95, 99, 101, 103, 111, 131, 133, 134, 135, 136, 139, 140, 143, 145, and155, or any combination thereof from SEQ ID NO: 2 or the correspondingamino acids of SEQ ID NO: 1 or 3. An examination of the crystalstructure of GH, e.g., hGH and its interactions with the GH, e.g., hGHreceptor indicates that the side chains of these amino acid residues arefully exposed to the solvent and the side chain of the native residuepoints out into the solvent.

A subset of exemplary sites for incorporation of one or morenon-naturally encoded amino acids include sites corresponding to 30, 74,103, or any combination thereof, from SEQ ID NO: 2 or the correspondingamino acids of SEQ ID NO: 1 or 3. Another subset of exemplary sites forincorporation of one or more non-naturally encoded amino acids includesites corresponding to 35, 92, 143, 145, or any combination thereof,from SEQ ID NO: 2 or the corresponding amino acids of SEQ ID NO: 1 or 3.A further subset of exemplary sites for incorporation of one or morenon-naturally encoded amino acids include sites corresponding to 35, 92,131, 134, 143, 145, or any combination thereof, from SEQ ID NO: 2 or thecorresponding amino acids of SEQ ID NO: 1 or 3. Still a further subsetof exemplary sites for incorporation of one or more non-naturallyencoded amino acids include sites corresponding to 30, 35, 74, 92, 103,145, or any combination thereof, from SEQ ID NO: 2 or the correspondingamino acids of SEQ ID NO: 1 or 3. Yet a further subset of exemplarysites for incorporation of one or more non-naturally encoded amino acidsinclude sites corresponding to 35, 92, 143, 145, or any combinationthereof, from SEQ ID NO: 2 or the corresponding amino acids of SEQ IDNO: 1 or 3. In certain embodiments, sites for incorporation of one ormore non-naturally encoded amino acids include a site corresponding to35 from SEQ ID NO: 2 or the corresponding amino acids of SEQ ID NO: 1 or3.

In some embodiments, at least one of the non-naturally encoded aminoacids incorporated into the GH, e.g., hGH, contains a carbonyl group,e.g., a ketone group. In certain embodiments, at least one of thenon-naturally encoded amino acids incorporated into the GH, e.g., hGH ispara-acetylphenylalanine. In some embodiments in which the GH, e.g., hGHcontains a plurality of non-naturally-encoded amino acids, more than oneof the non-naturally-encoded amino acids incorporated into the GH, e.g.,hGH is para-acetylphenylalanine. In some embodiments in which the GH,e.g., hGH contains a plurality of non-naturally-encoded amino acids,substantially all of the non-naturally-encoded amino acids incorporatedinto the GH, e.g., hGH are para-acetylphenylalanine.

In some embodiments, the non-naturally occurring amino acid is linked toa water soluble polymer at one or more positions, including but notlimited to, positions corresponding to: before position 1 (i.e. at theN-terminus), 1, 2, 3, 4, 5, 8, 9, 11, 12, 15, 16, 19, 22, 29, 30, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 52,55, 57, 59, 65, 66, 69, 70, 71, 74, 88, 91, 92, 94, 95, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 111, 112, 113, 115, 116,119, 120, 122, 123, 126, 127, 129, 130, 131, 132, 133, 134, 135, 136,137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150,151, 152, 153, 154, 155, 156, 158, 159, 161, 168, 172, 183, 184, 185,186, 187, 188, 189, 190, 191, 192 (i.e., at the carboxyl terminus of theprotein) (SEQ ID NO: 2 or the corresponding amino acids of SEQ ID NO: 1or 3). In some embodiments, the non-naturally occurring amino acid islinked to a water soluble polymer at positions including but not limitedto, positions corresponding to one or more of these positions: 30, 35,74, 92, 103, 143, 145 (SEQ ID NO: 2 or the corresponding amino acids ofSEQ ID NO: 1 or 3). In some embodiments, the non-naturally occurringamino acid is linked to a water soluble polymer at positions includingbut not limited to, positions corresponding to one or more of thesepositions: 35, 92, 143, 145 (SEQ ID NO: 2 or the corresponding aminoacids of SEQ ID NO: 1 or 3). In some embodiments, the non-naturallyoccurring amino acid is linked to a water soluble polymer at positionsincluding but not limited to, positions corresponding to one or more ofthese positions: 35, 92, 131, 134, 143, 145, or any combination thereof,from SEQ ID NO: 2 or the corresponding amino acids of SEQ ID NO: 1 or 3.In some embodiments, the non-naturally occurring amino acid is linked toa water soluble polymer at positions including but not limited to,positions corresponding to one or more of these positions: 30, 35, 74,92, 103, 145, or any combination thereof, from SEQ ID NO: 2 or thecorresponding amino acids of SEQ ID NO: 1 or 3. In some embodiments, thenon-naturally occurring amino acid is linked to a water soluble polymerat positions including but not limited to, positions corresponding toone or more of these positions: 35, 92, 143, 145, or any combinationthereof, from SEQ ID NO: 2 or the corresponding amino acids of SEQ IDNO: 1 or 3. In some embodiments, the non-naturally occurring amino acidis linked to a water-soluble polymer at a position corresponding to, butnot limited to, position 35 from SEQ ID NO: 2 or the corresponding aminoacids of SEQ ID NO: 1 or 3 is linked to a water-soluble polymer.

In some embodiments the water-soluble polymer(s) linked to the GH, e.g.,hGH, include one or more polyethylene glycol molecules (PEGs). Thepolymer, e.g., PEG, may be linear or branched. Typically, linearpolymers, e.g., PEGs, used in the invention can have a MW of about 0.1to about 100 kDa, or about 1 to about 60 kDa, or about 20 to about 40kDa, or about 30 kDa. Typically, branched polymers, e.g., PEGs, used inthe invention can have a MW of about 1 to about 100 kDa, or about 30 toabout 50 kDa, or about 40 kDa. Polymers such as PEGs are describedfurther herein. In certain embodiments, the linkage between the GH,e.g., hGH and the water-soluble polymer, e.g., PEG, is an oxime bond.

Certain embodiments of the invention encompass compositions that includea GH, e.g., hGH, linked to at least one water-soluble polymer by acovalent bond, where the covalent bond is an oxime bond. In someembodiments, the water-soluble polymer is a PEG, e.g., a linear PEG. Insome embodiments encompassing at least one linear PEG linked by an oximebond to a GH, e.g., hGH, the PEG can have a MW of about 0.1 to about 100kDa, or about 1 to about 60 kDa, or about 20 to about 40 kDa, or about30 kDa. In certain embodiments encompassing a linear PEG linked by anoxime bond to a GH, e.g., hGH, the PEG has a MW of about 30 kDa. In someembodiments encompassing at least one branched PEG linked by an oximebond to a GH, e.g., hGH, the PEG can have a MW of about 1 to about 100kDa or about 30 to about 50 kDa, or about 40 kDa. In certain embodimentsencompassing a branched PEG linked by an oxime bond to a GH, e.g., hGH,the PEG has a MW of about 40 kDa. In some embodiments, the GH is a GH,e.g., hGH and in certain of these embodiments, the GH, e.g., hGH has asequence that is at least about 80% identical to SEQ ID NO: 2; in someembodiments the GH, e.g., hGH has a sequence that is the sequence of SEQID NO: 2. In some embodiments, the GH, e.g., hGH, contains at least onenon-naturally-encoded amino acid; in some of these embodiments, at leastone oxime bond is between the non-naturally-encoded amino acid and atleast one water-soluble polymer. In some embodiments, thenon-naturally-encoded amino acid contains a carbonyl group, such as aketone group; in some embodiments, the non-naturally-encoded amino acidis para-acetylphenylalanine. In some embodiments, thepara-acetylphenylalanine is substituted at a position corresponding toposition 35 of SEQ ID NO: 2.

Thus, in some embodiments, the invention provides a GH, e.g., hGH,linked to at least one water-soluble polymer, e.g., a PEG, by a covalentbond, where the covalent bond is an oxime bond. In certain embodiments,the water-soluble polymer is a PEG and the PEG is a linear PEG. In theseembodiments, the linear PEG has a MW of about 0.1 to about 100 kDa, orabout 1 to about 60 kDa, or about 20 to about 40 kDa, or about 30 kDa.In certain embodiments encompassing a linear PEG linked by an oxime bondto a GH, e.g., hGH, the PEG has a MW of about 30 kDa. In certainembodiments, the water-soluble polymer is a PEG that is a branched PEG.In these embodiments, the branched PEG has a MW of about 1 to about 100kDa, or about 30 to about 50 kDa, or about 40 kDa. In certainembodiments encompassing a branched PEG linked by an oxime bond to a GH,e.g., hGH, the PEG has a MW of about 40 kDa.

In some embodiments, the invention provides a GH, e.g., hGH, where theGH, e.g., hGH contains a non-naturally encoded amino acid, where the GHis linked to at least one water-soluble polymer, e.g., a PEG, by acovalent bond, and where the covalent bond is an oxime bond between thenon-naturally encoded amino acid and the water-soluble polymer, e.g.,PEG. In some embodiments, the non-naturally-encoded amino acid isincorporated into the GH, e.g., hGH, at a position corresponding toposition 35 of SEQ ID NO: 2. In certain embodiments where thewater-soluble polymer is a PEG, the PEG is a linear PEG. In theseembodiments, the linear PEG has a MW of about 0.1 to about 100 kDa, orabout 1 to about 60 kDa, or about 20 to about 40 kDa, or about 30 kDa.In certain embodiments encompassing a linear PEG linked by an oxime bondto a GH, e.g., hGH, the PEG has a MW of about 30 kDa. In certainembodiments where the water-soluble polymer is a PEG, the PEG is abranched PEG. In these embodiments, the branched PEG has a MW of about 1to about 100 kDa, or about 30 to about 50 kDa, or about 40 kDa. Incertain embodiments encompassing a branched PEG linked by an oxime bondto a GH, e.g., hGH, the PEG has a MW of about 40 kDa.

In some embodiments, the invention provides a GH, e.g., hGH, where theGH, e.g., hGH contains a non-naturally encoded amino acid that is acarbonyl-containing non-naturally encoded amino acid, where the GH islinked to at least one water-soluble polymer, e.g., a PEG, by a covalentbond, and where the covalent bond is an oxime bond between thenon-naturally encoded carbonyl-containing amino acid and thewater-soluble polymer, e.g., PEG. In some embodiments, thenon-naturally-encoded carbonyl-containing amino acid is incorporatedinto the GH, e.g., hGH, at a position corresponding to position 35 ofSEQ ID NO: 2. In certain embodiments where the water-soluble polymer isa PEG, the PEG is a linear PEG. In these embodiments, the linear PEG hasa MW of about 0.1 to about 100 kDa, or about 1 to about 60 kDa, or about20 to about 40 kDa, or about 30 kDa. In certain embodiments encompassinga linear PEG linked by an oxime bond to a GH, e.g., hGH, the PEG has aMW of about 30 kDa. In certain embodiments where the water-solublepolymer is a PEG, the PEG is a branched PEG. In these embodiments, thebranched PEG has a MW of about 1 to about 100 kDa, or about 30 to about50 kDa, or about 40 kDa. In certain embodiments encompassing a branchedPEG linked by an oxime bond to a GH, e.g., hGH, the PEG has a MW ofabout 40 kDa.

In some embodiments, the invention provides a GH, e.g., hGH, thatcontains a non-naturally encoded amino acid that includes a ketonegroup, where the GH is linked to at least one water-soluble polymer,e.g., a PEG, by a covalent bond, and where the covalent bond is an oximebond between the non-naturally encoded amino acid containing a ketonegroup and the water-soluble polymer, e.g., PEG. In some embodiments, thenon-naturally-encoded amino acid containing a ketone group isincorporated into the GH, e.g., hGH, at a position corresponding toposition 35 of SEQ ID NO: 2. In certain embodiments where thewater-soluble polymer is a PEG, the PEG is a linear PEG. In theseembodiments, the linear PEG has a MW of about 0.1 to about 100 kDa, orabout 1 to about 60 kDa, or about 20 to about 40 kDa, or about 30 kDa.In certain embodiments encompassing a linear PEG linked by an oxime bondto a GH, e.g., hGH, the PEG has a MW of about 30 kDa. In certainembodiments where the water-soluble polymer is a PEG, the PEG is abranched PEG. In these embodiments, the branched PEG has a MW of about 1to about 100 kDa, or about 30 to about 50 kDa, or about 40 kDa. Incertain embodiments encompassing a branched PEG linked by an oxime bondto a GH, e.g., hGH, the PEG has a MW of about 40 kDa.

In some embodiments, the invention provides a GH, e.g., hGH, thatcontains a non-naturally encoded amino acid that is apara-acetylphenylalanine, where the GH linked to at least onewater-soluble polymer, e.g., a PEG, by a covalent bond, and where thecovalent bond is an oxime bond between the para-acetylphenylalanine andthe water-soluble polymer, e.g., PEG. In some embodiments, thepara-acetylphenylalanine is incorporated into the GH, e.g., hGH, at aposition corresponding to position 35 of SEQ ID NO: 2. In certainembodiments where the water-soluble polymer is a PEG, the PEG is alinear PEG. In these embodiments, the linear PEG has a MW of about 0.1to about 100 kDa, or about 1 to about 60 kDa, or about 20 to about 40kDa, or about 30 kDa. In certain embodiments encompassing a linear PEGlinked by an oxime bond to a GH, e.g., hGH, the PEG has a MW of about 30kDa. In certain embodiments where the water-soluble polymer is a PEG,the PEG is a branched PEG. In these embodiments, the branched PEG has aMW of about 1 to about 100 kDa, or about 30 to about 50 kDa, or about 40kDa. In certain embodiments encompassing a branched PEG linked by anoxime bond to a GH, e.g., hGH, the PEG has a MW of about 40 kDa.

In certain embodiments the invention provides a GH, e.g., hGH thatincludes SEQ ID NO: 2, and where the GH, e.g., hGH is substituted at aposition corresponding to position 35 of SEQ ID NO: 2 with apara-acetylphenylalanine that is linked by an oxime linkage to a linearPEG of MW of about 30 kDa.

In some embodiments, the invention provides a hormone composition thatincludes a GH, e.g., hGH, linked via an oxime bond to at least one PEG,e.g., a linear PEG, where the GH, e.g., hGH comprises the amino acidsequence of SEQ ID NO: 2, and where the GH, e.g., hGH contains at leastone non-naturally-encoded amino acid substituted at one or morepositions including, but not limited to, positions corresponding to:before position 1 (i.e. at the N-terminus), 1, 2, 3, 4, 5, 8, 9, 11, 12,15, 16, 19, 22, 29, 30, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 52, 55, 57, 59, 65, 66, 69, 70, 71, 74, 88, 91,92, 94, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,109, 111, 112, 113, 115, 116, 119, 120, 122, 123, 126, 127, 129, 130,131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144,145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 158, 159,161, 168, 172, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192 (i.e.,at the carboxyl terminus of the protein) (SEQ ID NO: 2 or thecorresponding amino acids of SEQ ID NO: 1 or 3). In some embodiments,the invention provides a hormone composition that includes a GH, e.g.,hGH, linked via an oxime bond to at least one PEG, e.g., a linear PEG,where the GH, e.g., hGH comprises the amino acid sequence of SEQ ID NO:2, and where the GH, e.g., hGH contains at least onenon-naturally-encoded amino acid substituted at one or more positionsincluding, but not limited to, positions corresponding to: 30, 35, 74,92, 103, 143, 145 (SEQ ID NO: 2 or the corresponding amino acids of SEQID NO: 1 or 3). In some embodiments, the invention provides a hormonecomposition that includes a GH, e.g., hGH, linked via an oxime bond toat least one PEG, e.g., a linear PEG, where the GH, e.g., hGH comprisesthe amino acid sequence of SEQ ID NO: 2, and where the GH, e.g., hGHcontains at least one non-naturally-encoded amino acid substituted atone or more positions including, but not limited to, positionscorresponding to: 35, 92, 143, 145 (SEQ ID NO: 2 or the correspondingamino acids of SEQ ID NO: 1 or 3). In some embodiments, the inventionprovides a hormone composition that includes a GH, e.g., hGH, linked viaan oxime bond to at least one PEG, e.g., a linear PEG, where the GH,e.g., hGH comprises the amino acid sequence of SEQ ID NO: 2, and wherethe GH, e.g., hGH contains at least one non-naturally-encoded amino acidsubstituted at one or more positions including, but not limited to,positions corresponding to: 35, 92, 131, 134, 143, 145, or anycombination thereof, from SEQ ID NO: 2 or the corresponding amino acidsof SEQ ID NO: 1 or 3. In some embodiments, the invention provides ahormone composition that includes a GH, e.g., hGH, linked via an oximebond to at least one PEG, e.g., a linear PEG, where the GH, e.g., hGHcomprises the amino acid sequence of SEQ ID NO: 2, and where the GH,e.g., hGH contains at least one non-naturally-encoded amino acidsubstituted at one or more positions including, but not limited to,positions corresponding to: 30, 35, 74, 92, 103, 145, or any combinationthereof, from SEQ ID NO: 2 or the corresponding amino acids of SEQ IDNO: 1 or 3. In some embodiments, the invention provides a hormonecomposition that includes a GH, e.g., hGH, linked via an oxime bond toat least one PEG, e.g., a linear PEG, where the GH, e.g., hGH comprisesthe amino acid sequence of SEQ ID NO: 2, and where the GH, e.g., hGHcontains at least one non-naturally-encoded amino acid substituted atone or more positions including, but not limited to, positionscorresponding to: 35, 92, 143, 145, or any combination thereof, from SEQID NO: 2 or the corresponding amino acids of SEQ ID NO: 1 or 3. In someembodiments, the invention provides a hormone composition that includesa GH, e.g., hGH, linked via an oxime bond to at least one PEG, e.g., alinear PEG, where the GH, e.g., hGH comprises the amino acid sequence ofSEQ ID NO: 2, and where the GH, e.g., hGH contains at least onenon-naturally-encoded amino acid substituted at one or more positionsincluding, but not limited to, positions corresponding to position 35from SEQ ID NO: 2 or the corresponding amino acids of SEQ ID NO: 1 or 3.In embodiments in which the PEG is a linear PEG, the PEG can have a MWof about 0.1 to about 100 kDa, or about 1 to about 60 kDa, or about 20to about 40 kDa, or about 30 kDa.

In some embodiments, the invention provides a hormone composition thatincludes a GH, e.g., hGH, linked via an oxime bond to at least one PEG,e.g., a linear PEG, where the GH, e.g., hGH includes the amino acidsequence of SEQ ID NO: 2, and where the GH, e.g., hGH contains at leastone non-naturally-encoded amino acid that is a para-acetylphenylalaninesubstituted at one or more positions including, but not limited to,positions corresponding to: before position 1 (i.e. at the N-terminus),1, 2, 3, 4, 5, 8, 9, 11, 12, 15, 16, 19, 22, 29, 30, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 52, 55, 57, 59, 65,66, 69, 70, 71, 74, 88, 91, 92, 94, 95, 97, 98, 99, 100, 101, 102, 103,104, 105, 106, 107, 108, 109, 111, 112, 113, 115, 116, 119, 120, 122,123, 126, 127, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139,140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153,154, 155, 156, 158, 159, 161, 168, 172, 183, 184, 185, 186, 187, 188,189, 190, 191, 192 (i.e., at the carboxyl terminus of the protein) (SEQID NO: 2 or the corresponding amino acids of SEQ ID NO: 1 or 3). In someembodiments, the invention provides a hormone composition that includesa GH, e.g., hGH, linked via an oxime bond to at least one PEG, e.g., alinear PEG, where the GH, e.g., hGH comprises the amino acid sequence ofSEQ ID NO: 2, and where the GH, e.g., hGH contains at least onenon-naturally-encoded amino acid that is a para-acetylphenylalaninesubstituted at one or more positions including, but not limited to,positions corresponding to: 30, 35, 74, 92, 103, 143, 145 (SEQ ID NO: 2or the corresponding amino acids of SEQ ID NO: 1 or 3). In someembodiments, the invention provides a hormone composition that includesa GH, e.g., hGH, linked via an oxime bond to at least one PEG, e.g., alinear PEG, where the GH, e.g., hGH comprises the amino acid sequence ofSEQ ID NO: 2, and where the GH, e.g., hGH contains at least onenon-naturally-encoded amino acid that is a para-acetylphenylalaninesubstituted at one or more positions including, but not limited to,positions corresponding to: 35, 92, 143, 145 (SEQ ID NO: 2 or thecorresponding amino acids of SEQ ID NO: 1 or 3). In some embodiments,the invention provides a hormone composition that includes a GH, e.g.,hGH, linked via an oxime bond to at least one PEG, e.g., a linear PEG,where the GH, e.g., hGH comprises the amino acid sequence of SEQ ID NO:2, and where the GH, e.g., hGH contains at least onenon-naturally-encoded amino acid that is a para-acetylphenylalaninesubstituted at one or more positions including, but not limited to,positions corresponding to: 35, 92, 131, 134, 143, 145, or anycombination thereof, from SEQ ID NO: 2 or the corresponding amino acidsof SEQ ID NO: 1 or 3. In some embodiments, the invention provides ahormone composition that includes a GH, e.g., hGH, linked via an oximebond to at least one PEG, e.g., a linear PEG, where the GH, e.g., hGHcomprises the amino acid sequence of SEQ ID NO: 2, and where the GH,e.g., hGH contains at least one non-naturally-encoded amino acid that isa para-acetylphenylalanine substituted at one or more positionsincluding, but not limited to, positions corresponding to: 30, 35, 74,92, 103, 145, or any combination thereof, from SEQ ID NO: 2 or thecorresponding amino acids of SEQ ID NO: 1 or 3. In some embodiments, theinvention provides a hormone composition that includes a GH, e.g., hGH,linked via an oxime bond to at least one PEG, e.g., a linear PEG, wherethe GH, e.g., hGH comprises the amino acid sequence of SEQ ID NO: 2, andwhere the GH, e.g., hGH contains at least one non-naturally-encodedamino acid that is a para-acetylphenylalanine substituted at one or morepositions including, but not limited to, positions corresponding to: 35,92, 143, 145, or any combination thereof, from SEQ ID NO: 2 or thecorresponding amino acids of SEQ ID NO: 1 or 3. In some embodiments, theinvention provides a hormone composition that includes a GH, e.g., hGH,linked via an oxime bond to at least one PEG, e.g., a linear PEG, wherethe GH, e.g., hGH comprises the amino acid sequence of SEQ ID NO: 2, andwhere the GH, e.g., hGH contains at least one non-naturally-encodedamino acid that is a para-acetylphenylalanine substituted at one or morepositions including, but not limited to, positions corresponding toposition 35 from SEQ ID NO: 2 or the corresponding amino acids of SEQ IDNO: 1 or 3. In embodiments in which the PEG is a linear PEG, the PEG canhave a MW of about 0.1 to about 100 kDa, or about 1 to about 60 kDa, orabout 20 to about 40 kDa, or about 30 kDa.

In some embodiments, the invention provides a GH, e.g., hGH, where theGH, e.g., hGH contains at least one non-naturally encoded amino acid,where the GH is linked to a plurality of water-soluble polymers, e.g., aplurality of PEGs, by covalent bonds, where one or more of the covalentbond is an oxime bond between at least one of the non-naturally encodedamino acid and the water-soluble polymer, e.g., PEG. The GH, e.g., hGH,may be linked to about 2-100 water-soluble polymers, e.g., PEGs, orabout 2-50 water-soluble polymers, e.g., PEGs, or about 2-25water-soluble polymers, e.g., PEGs, or about 2-10 water-solublepolymers, e.g., PEGs, or about 2-5 water-soluble polymers, e.g., PEGs,or about 5-100 water-soluble polymers, e.g., PEGs, or about 5-50water-soluble polymers, e.g., PEGs, or about 5-25 water-solublepolymers, e.g., PEGs, or about 5-10 water-soluble polymers, e.g., PEGs,or about 10-100 water-soluble polymers, e.g., PEGs, or about 10-50water-soluble polymers, e.g., PEGs, or about 10-20 water-solublepolymers, e.g., PEGs, or about 20-100 water-soluble polymers, e.g.,PEGs, or about 20-50 water-soluble polymers, e.g., PEGs, or about 50-100water-soluble polymers, e.g., PEGs. The one or morenon-naturally-encoded amino acids may be incorporated into the GH, e.g.,hGH, at any position described herein. In some embodiments, at least onenon-naturally-encoded amino acid is incorporated into the GH, e.g., hGH,at a position corresponding to position 35 of SEQ ID NO: 2. In someembodiments, the non-naturally encoded amino acids include at least onenon-naturally encoded amino acid that is a carbonyl-containingnon-naturally encoded amino acid, e.g., a ketone-containingnon-naturally encoded amino acid such as a para-acetylphenylalanine. Insome embodiments, the GH, e.g., hGH, includes apara-acetylphenylalanine. In some embodiments, thepara-acetylphenylalanine is incorporated into the GH, e.g., hGH, at aposition corresponding to position 35 of SEQ ID NO: 2, where thepara-acetylphenylalanine is linked to one of the polymers, e.g., one ofthe PEGs, by an oxime bond. In some embodiments, at least one of thewater-soluble polymers, e.g., PEGs, is linked to the GH, e.g., hGH, by acovalent bond to at least one of the non-naturally-encoded amino acids.In some embodiments, the covalent bond is an oxime bond. In someembodiments, a plurality of the water-soluble polymers, e.g., PEGs, arelinked to the GH, e.g., hGH, by covalent bonds to a plurality of thenon-naturally-encoded amino acids. In some embodiments, at least one thecovalent bonds is an oxime bond; in some embodiments, a plurality of thecovalent bonds are oxime bonds; in some embodiments, substantially allof the bonds are oxime bonds. The plurality of water-soluble polymers,e.g., PEG, may be linear, branched, or any combination thereof. Inembodiments that incorporate one or more linear PEGs, the linear PEGshave a MW of about 0.1 to about 100 kDa, or about 1 to about 60 kDa, orabout 20 to about 40 kDa, or about 30 kDa. In embodiments thatincorporate one or more branched PEGs, the branched PEGs have a MW ofabout 1 to about 100 kDa, or about 30 to about 50 kDa, or about 40 kDa.It will be appreciated that embodiments employing a plurality ofwater-soluble polymers, e.g., PEGs, will, in general, employ suchpolymers at lower MWs than embodiments in which a single PEG is used.Thus, in some embodiments, the overall MW of the plurality of PEGs isabout 0.1-500 kDa, or about 0.1-200 kDa, or about 0.1-100 kDa, or about1-1000 kDa, or about 1-500 kDa, or about 1-200 kDa, or about 1-100 kDa,or about 10-1000 kDa, or about 10-500 kDa, or about 10-200 kDa, or about10-100 kDa, or about 10-50 kDa, or about 20-1000 kDa, or about 20-500kDa, or about 20-200 kDa, or about 20-100 kDa, or about 20-80 kDa, about20-60 kDa, about 5-100 kDa, about 5-50 kDa, or about 5-20 kDa.

Human GH antagonists include, but are not limited to, those withsubstitutions at: 1, 2, 3, 4, 5, 8, 9, 11, 12, 15, 16, 19, 22, 103, 109,112, 113, 115, 116, 119, 120, 123, and 127 or an addition at position 1(i.e., at the N-terminus), or any combination thereof (SEQ ID NO:2, orthe corresponding amino acid in SEQ ID NO: 1, 3, or any other GHsequence).

A wide variety of non-naturally encoded amino acids can be substitutedfor, or incorporated into, a given position in a GH, e.g., hGHpolypeptide. In general, a particular non-naturally encoded amino acidis selected for incorporation based on an examination of the threedimensional crystal structure of a GH, e.g., hGH polypeptide with itsreceptor, a preference for conservative substitutions (i.e., aryl-basednon-naturally encoded amino acids, such as p-acetylphenylalanine orO-propargyltyrosine substituting for Phe, Tyr or Trp), and the specificconjugation chemistry that one desires to introduce into the GH, e.g.,hGH polypeptide (e.g., the introduction of 4-azidophenylalanine if onewants to effect a Huisgen [3+2]cycloaddition with a water solublepolymer bearing an alkyne moiety or a amide bond formation with a watersoluble polymer that bears an aryl ester that, in turn, incorporates aphosphine moiety).

In one embodiment, the method further includes incorporating into theprotein the unnatural amino acid, where the unnatural amino acidcomprises a first reactive group; and contacting the protein with amolecule (including but not limited to, a label, a dye, a polymer, awater-soluble polymer, a derivative of polyethylene glycol, aphotocrosslinker, a radionuclide, a cytotoxic compound, a drug, anaffinity label, a photoaffinity label, a reactive compound, a resin, asecond protein or polypeptide or polypeptide analog, an antibody orantibody fragment, a metal chelator, a cofactor, a fatty acid, acarbohydrate, a polynucleotide, a DNA, a RNA, an antisensepolynucleotide, a saccharide, water-soluble dendrimer, a cyclodextrin,an inhibitory ribonucleic acid, a biomaterial, a nanoparticle, a spinlabel, a fluorophore, a metal-containing moiety, a radioactive moiety, anovel functional group, a group that covalently or noncovalentlyinteracts with other molecules, a photocaged moiety, an actinicradiation excitable moiety, a photoisomerizable moiety, biotin, aderivative of biotin, a biotin analogue, a moiety incorporating a heavyatom, a chemically cleavable group, a photocleavable group, an elongatedside chain, a carbon-linked sugar, a redox-active agent, an aminothioacid, a toxic moiety, an isotopically labeled moiety, a biophysicalprobe, a phosphorescent group, a chemiluminescent group, an electrondense group, a magnetic group, an intercalating group, a chromophore, anenergy transfer agent, a biologically active agent, a detectable label,a small molecule, a quantum dot, a nanotransmitter, a radionucleotide, aradiotransmitter, a neutron-capture agent, or any combination of theabove, or any other desirable compound or substance) that comprises asecond reactive group. The first reactive group reacts with the secondreactive group to attach the molecule to the unnatural amino acidthrough a [3+2]cycloaddition. In one embodiment, the first reactivegroup is an alkynyl or azido moiety and the second reactive group is anazido or alkynyl moiety. For example, the first reactive group is thealkynyl moiety (including but not limited to, in unnatural amino acidp-propargyloxyphenylalanine) and the second reactive group is the azidomoiety. In another example, the first reactive group is the azido moiety(including but not limited to, in the unnatural amino acidp-azido-L-phenylalanine) and the second reactive group is the alkynylmoiety.

In some cases, the non-naturally encoded amino acid substitution(s) willbe combined with other additions, substitutions or deletions within theGH, e.g., hGH polypeptide to affect other biological traits of the GH,e.g., hGH polypeptide. In some cases, the other additions, substitutionsor deletions may increase the stability (including but not limited to,resistance to proteolytic degradation) of the GH, e.g., hGH polypeptideor increase affinity of the GH, e.g., hGH polypeptide for its receptor.In some embodiments, the GH, e.g., hGH polypeptide comprises an aminoacid substitution selected from the group consisting of F10A, F10H,F10I; M14W, M14Q, M14G; H18D; H21N; G120A; R167N; D171S; E174S; F176Y,1179T or any combination thereof in SEQ ID NO: 2. In some cases, theother additions, substitutions or deletions may increase the solubility(including but not limited to, when expressed in E. coli or other hostcells) of the GH, e.g., hGH polypeptide. In some embodiments additions,substitutions or deletions may increase the polypeptide solubilityfollowing expression in E. coli or other recombinant host cells. In someembodiments sites are selected for substitution with a naturally encodedor non-natural amino acid in addition to another site for incorporationof a non-natural amino acid that results in increasing the polypeptidesolubility following expression in E. coli or other recombinant hostcells. In some embodiments, the GH, e.g., hGH polypeptides compriseanother addition, substitution or deletion that modulates affinity forthe GH, e.g., hGH polypeptide receptor, modulates (including but notlimited to, increases or decreases) receptor dimerization, stabilizesreceptor dimers, modulates circulating half-life, modulates release orbio-availability, facilitates purification, or improves or alters aparticular route of administration. For instance, in addition tointroducing one or more non-naturally encoded amino acids as set forthherein, one or more of the following substitutions are introduced: F10A,F10H or F10I; M14W, M14Q, or M14G; H18D; H21N; R167N; D171S; E174S;F176Y and 1179T to increase the affinity of the GH, e.g., hGH variantfor its receptor. Similarly, GH, e.g., hGH polypeptides can comprisechemical or enzyme cleavage sequences, protease cleavage sequences,reactive groups, antibody-binding domains (including but not limited to,FLAG or poly-His) or other affinity based sequences (including, but notlimited to, FLAG, poly-His, GST, etc.) or linked molecules (including,but not limited to, biotin) that improve detection (including, but notlimited to, GFP), purification, transport through tissues or cellmembranes, prodrug release or activation, hGH size reduction, or othertraits of the polypeptide.

In some embodiments, the substitution of a non-naturally encoded aminoacid generates an GH, e.g., hGH antagonist. A subset of exemplary sitesfor incorporation of one or more non-naturally encoded amino acidinclude: 1, 2, 3, 4, 5, 8, 9, 11, 12, 15, 16, 19, 22, 103, 109, 112,113, 115, 116, 119, 120, 123, 127, or an addition before position 1 (SEQID NO: 2, or the corresponding amino acid in SEQ ID NO: 1, 3, or anyother GH sequence). In some embodiments, GH, e.g., hGH antagonistscomprise at least one substitution in the regions 1-5 (N-terminus), 6-33(A helix), 34-74 (region between A helix and B helix, the A-B loop),75-96 (B helix), 97-105 (region between B helix and C helix, the B-Cloop), 106-129 (C helix), 130-153 (region between C helix and D helix,the C-D loop), 154-183 (D helix), 184-191 (C-terminus) that cause GH toact as an antagonist. In other embodiments, the exemplary sites ofincorporation of a non-naturally encoded amino acid include residueswithin the amino terminal region of helix A and a portion of helix C. Inanother embodiment, substitution of G120 with a non-naturally encodedamino acid such as p-azido-L-phenyalanine or O-propargyl-L-tyrosine. Inother embodiments, the above-listed substitutions are combined withadditional substitutions that cause the GH, e.g., hGH polypeptide to bean GH, e.g., hGH antagonist. For instance, a non-naturally encoded aminoacid is substituted at one of the positions identified herein and asimultaneous substitution is introduced at G120 (e.g., G120R, G120K,G120W, G120Y, G120F, or G120E). In some embodiments, the GH, e.g., hGHantagonist comprises a non-naturally encoded amino acid linked to awater soluble polymer that is present in a receptor binding region ofthe GH, e.g., hGH molecule.

In some cases, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids aresubstituted with one or more non-naturally-encoded amino acids. In somecases, the GH, e.g., hGH polypeptide further includes 1, 2, 3, 4, 5, 6,7, 8, 9, 10, or more substitutions of one or more non-naturally encodedamino acids for naturally-occurring amino acids. For example, in someembodiments, one or more residues in the following regions of GH, e.g.,hGH are substituted with one or more non-naturally encoded amino acids:1-5 (N-terminus); 32-46 (N-terminal end of the A-B loop); 97-105 (B-Cloop); and 132-149 (C-D loop); and 184-191 (C-terminus). In someembodiments, one or more residues in the following regions of GH, e.g.,hGH are substituted with one or more non-naturally encoded amino acids:1-5 (N-terminus), 6-33 (A helix), 34-74 (region between A helix and Bhelix, the A-B loop), 75-96 (B helix), 97-105 (region between B helixand C helix, the B-C loop), 106-129 (C helix), 130-153 (region between Chelix and D helix, the C-D loop), 154-183 (D helix), 184-191(C-terminus). In some cases, the one or more non-naturally encodedresidues are linked to one or more lower molecular weight linear orbranched PEGs (approximately ˜5-20 kDa in mass or less), therebyenhancing binding affinity and comparable serum half-life relative tothe species attached to a single, higher molecular weight PEG.

In some embodiments, up to two of the following residues of GH, e.g.,hGH are substituted with one or more non-naturally-encoded amino acidsat position: 29, 30, 33, 34, 35, 37, 39, 40, 49, 57, 59, 66, 69, 70, 71,74, 88, 91, 92, 94, 95, 98, 99, 101, 103, 107, 108, 111, 122, 126, 129,130, 131, 133, 134, 135, 136, 137, 139, 140, 141, 142, 143, 145, 147,154, 155, 156, 159, 183, 186, and 187. In some cases, any of thefollowing pairs of substitutions are made: K38X* and K140X*; K41X* andK145X*; Y35X* and E88X*; Y35X* and F92X*; Y35X* and Y143X*; F92X* andY143X* wherein X* represents a non-naturally encoded amino acid.Preferred sites for incorporation of two or more non-naturally encodedamino acids include combinations of the following residues: 29, 33, 35,37, 39, 49, 57, 69, 70, 71, 74, 88, 91, 92, 94, 95, 98, 99, 101, 103,107, 108, 111, 129, 130, 131, 133, 134, 135, 136, 137, 139, 140, 141,142, 143, 145, 147, 154, 155, 156, 186, and 187. Particularly preferredsites for incorporation of two or more non-naturally encoded amino acidsinclude combinations of the following residues: 35, 88, 91, 92, 94, 95,99, 101, 103, 111, 131, 133, 134, 135, 136, 139, 140, 143, 145, and 155.

Preferred sites for incorporation in GH, e.g., hGH of two or morenon-naturally encoded amino acids include combinations of the followingresidues: before position 1 (i.e. at the N-terminus), 1, 2, 3, 4, 5, 8,9, 11, 12, 15, 16, 19, 22, 29, 30, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 52, 55, 57, 59, 65, 66, 69, 70, 71,74, 88, 91, 92, 94, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106,107, 108, 109, 111, 112, 113, 115, 116, 119, 120, 122, 123, 126, 127,129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,158, 159, 161, 168, 172, 183, 184, 185, 186, 187, 188, 189, 190, 191,192 (i.e. at the carboxyl terminus of the protein) or any combinationthereof from SEQ ID NO: 2.

VIII. Measurement of hGH Polypeptide Activity and Affinity of hGHPolypeptide for the hGH Polypeptide Receptor

Activity of the hGH may be measured using any of several techniquesknown in the art, including, but not limited to, cell binding assays orpSTAT5 assay on IM9 cells. To assess the biological activity of modifiedhGH polypeptides, assays monitoring the interaction between hGH and itsreceptor may be used. For example, an assay measuring tyrosinephosphorylation of a signal transducer and activator of transcriptionfamily member, STAT5, in the human IM-9 lymphocyte cell line (ATCC,Manassas, Va.) may be used. See, e.g., Silva et al., Mol. Endocrinol.(1996) 10(5):508-518. The IM-9 cells were starved overnight in assaymedia (phenol-red free RPMI, 10 mM Hepes, 1% heat inactivatedcharcoal/dextran treated FBS, sodium pyruvate, penicillin andstreptomycin) before stimulation with a 12-point dose range of hGHpolypeptides for 10 min at 37° C. Stimulated cells were fixed with 1%formaldehyde before permeabilization with 90% ice-cold methanol for 1hour on ice. The level of STAT5 phosphorylation was detected byintra-cellular staining with a primary phospho-STAT5 antibody (CellSignaling Technology, Beverly, Mass.) at room temperature for 30 minfollowed by a PE-conjugated secondary antibody. Sample acquisition wasperformed on the FACS Array with acquired data analyzed on the Flowjosoftware (Tree Star Inc., Ashland, Oreg.). EC₅₀ values were derived fromdose response curves plotted with mean fluorescent intensity (MFI)against protein concentration utilizing SigmaPlot.

Alternatively, proliferation studies with BrdU may be done in a cellline such as BAF3 stably transfected with rat growth hormone receptor.Serum starved rat growth hormone receptor, GHR, (L43R) expressing BAF3cell line, 2E2-2B12-F4, were plated at a density of 5×10⁴ cells/well ina 96-well plate. Cells were activated with a 12-point dose range of hGHproteins and labeled at the same time with 50 uM BrdU (Sigma, St. Louis,Mo.). After 48 hours in culture, cells were fixed/permeabilized with 100ul of BD cytofix/cytoperm solution (BD Biosciences) for 30 min at roomtemperature. To expose BrdU epitopes, fixed/permeablilized cells weretreated with 30 ug/well of DNase (Sigma) for 1 hour at 37° C.Immunofluorescent staining with APC-conjugated anti-BrdU antibody (BDBiosciences) enabled sample analysis on the FACS Array.

The hGH receptor can be prepared as described in McFarland et al.,Science, 245: 494-499 (1989) and Leung, D., et al., Nature, 330:537-543(1987). hGH polypeptide activity can be determined using standard orknown in vitro or in vivo assays. For example, cell lines thatproliferate in the presence of hGH (e.g., a cell line expressing the hGHreceptor or a lactogenic receptor) can be used to monitor hGH receptorbinding. See, e.g., Clark, R., et al., J. Biol. Chem. 271(36):21969(1996); Wada, et al., Mol. Endocrinol. 12:146-156 (1998); Gout, P. W.,et al. Cancer Res. 40, 2433-2436 (1980); WO 99/03887. For anon-PEGylated or PEGYlated hGH polypeptide comprising a non-naturalamino acid, the affinity of the hormone for its receptor can be measuredby using a BIAcore™ biosensor (GE Healthcare). See, e.g., U.S. Pat. No.5,849,535; Spencer, S. A., et al., J. Biol. Chem., 263:7862-7867 (1988).In vivo animal models for testing hGH activity include those describedin, e.g., Clark et al., J. Biol. Chem. 271(36):21969-21977 (1996).Assays for dimerization capability of hGH polypeptides comprising one ormore non-naturally encoded amino acids can be conducted as described inCunningham, B., et al., Science, 254:821-825 (1991) and Fuh, G., et al.,Science, 256:1677-1680 (1992). All references and patents cited areincorporated by reference herein. The above compilation of referencesfor assay methodologies is not exhaustive, and those of ordinary skillin the art will recognize other assays useful for testing for thedesired end result.

IX. Measurement of Potency, Functional In Vivo Half-Life, andPharmacokinetic Parameters

An important aspect of the invention is the prolonged biologicalhalf-life that is obtained by construction of the hGH polypeptide withor without conjugation of the polypeptide to a water soluble polymermoiety. The rapid decrease of hGH polypeptide serum concentrations hasmade it important to evaluate biological responses to treatment withconjugated and non-conjugated hGH polypeptide and variants thereof. Theconjugated and non-conjugated hGH polypeptide and variants thereof ofthe present invention may have prolonged serum half-lives also aftersubcutaneous or i.v. administration, making it possible to measure by,e.g. ELISA method or by a primary screening assay. ELISA or RIA kitsfrom either BioSource International (Camarillo, Calif.) or DiagnosticSystems Laboratories (Webster, Tex.) may be used. Measurement of in vivobiological half-life is carried out as described herein.

The potency and functional in vivo half-life of an hGH polypeptidecomprising a non-naturally encoded amino acid can be determinedaccording to the protocol described in Clark, R., et al., J. Biol. Chem.271(36): 21969-21977 (1996).

Pharmacokinetic parameters for a hGH polypeptide comprising anon-naturally encoded amino acid can be evaluated in normalSprague-Dawley male rats (N=5 animals per treatment group). Animals willreceive either a single dose of 25 ug/rat iv or 50 ug/rat sc, andapproximately 5-7 blood samples will be taken according to a pre-definedtime course, generally covering about 6 hours for a hGH polypeptidecomprising a non-naturally encoded amino acid not conjugated to a watersoluble polymer and about 4 days for a hGH polypeptide comprising anon-naturally encoded amino acid and conjugated to a water solublepolymer. Pharmacokinetic data for hGH polypeptides is well-studied inseveral species and can be compared directly to the data obtained forhGH polypeptides comprising a non-naturally encoded amino acid. SeeMordenti J., et al., Pharm. Res. 8(11):1351-59 (1991) for studiesrelated to hGH.

Pharmacokinetic parameters can also be evaluated in a primate, e.g.,cynomolgus monkeys. Typically, a single injection is administered eithersubcutaneously or intravenously, and serum hGH levels are monitored overtime.

The specific activity of hGH polypeptides in accordance with thisinvention can be determined by various assays known in the art. Thebiological activity of the hGH polypeptide muteins, or fragmentsthereof, obtained and purified in accordance with this invention can betested by methods described or referenced herein or known to those ofordinary skill in the art.

X. Therapeutic Uses of hGH Polypeptides

The hGH agonist polypeptides may be useful, for example, for treatinggrowth deficiency, immune disorders, and for stimulating heart function.Individuals with growth deficiencies include, e.g., individuals withTurner's Syndrome, GH-deficient individuals (including children),children who experience a slowing or retardation in their normal growthcurve about 2-3 years before their growth plate closes (sometimes knownas “short normal children”), and individuals where the insulin-likegrowth factor-I (IGF-I) response to GH has been blocked chemically(i.e., by glucocorticoid treatment) or by a natural condition such as inadult patients where the IGF-I response to GH is naturally reduced. ThehGH polypeptides of the invention may be useful for treating individualswith the following conditions: pediatric growth hormone deficiency,idiopathic short stature, adult growth hormone deficiency of childhoodonset, adult growth hormone deficiency of adult onset, or secondarygrowth hormone deficiency. Adults diagnosed with growth hormonedeficiency in adulthood may have had a pituitary tumor or radiation.Conditions including but not limited to, metabolic syndrome, headinjury, obesity, osteoporosis, or depression may result in growthhormone deficiency-like symptoms in adults.

An agonist hGH variant may act to stimulate the immune system of amammal by increasing its immune function, whether the increase is due toantibody mediation or cell mediation, and whether the immune system isendogenous to the host treated with the hGH polypeptide or istransplanted from a donor to the host recipient given the hGHpolypeptide (as in bone marrow transplants). “Immune disorders” includeany condition in which the immune system of an individual has a reducedantibody or cellular response to antigens than normal, including thoseindividuals with small spleens with reduced immunity due to drug (e.g.,chemotherapeutic) treatments. Examples individuals with immune disordersinclude, e.g., elderly patients, individuals undergoing chemotherapy orradiation therapy, individuals recovering from a major illness, or aboutto undergo surgery, individuals with AIDS, Patients with congenital andacquired B-cell deficiencies such as hypogammaglobulinemia, commonvaried agammaglobulinemia, and selective immunoglobulin deficiencies(e.g., IgA deficiency, patients infected with a virus such as rabieswith an incubation time shorter than the immune response of the patient;and individuals with hereditary disorders such as diGeorge syndrome.

hGH antagonist polypeptides may be useful for the treatment of gigantismand acromegaly, diabetes and complications (diabetic retinopathy,diabetic neuropathy) arising from diabetes, vascular eye diseases (e.g.,involving proliferative neovascularization), nephropathy, andGH-responsive malignancies. Vascular eye diseases include, e.g.,retinopathy (caused by, e.g., pre-maturity or sickle cell anemia) andmacular degeneration. GH-responsive malignancies include, e.g., Wilm'stumor, sarcomas (e.g., osteogenic sarcoma), breast, colon, prostate, andthyroid cancer, and cancers of tissues that express GH receptor mRNA(i.e., placenta, thymus, brain, salivary gland, prostate, bone marrow,skeletal muscle, trachea, spinal cord, retina, lymph node and fromBurkitt's lymphoma, colorectal carcinoma, lung carcinoma, lymphoblasticleukemia, and melanoma).

The GH, e.g., hGH agonist polypeptides of the invention may be useful,for example, for treating chronic renal failure, growth failureassociated with chronic renal insufficiency (CR1), short statureassociated with Turner Syndrome, pediatric Prader-Willi Syndrome (PWS),HIV patients with wasting or cachexia, children born small forgestational age (SGA), obesity, and osteoporosis.

hGH polypeptides of the invention, including PEGylated hGH, may beadministered by any conventional route suitable for proteins orpeptides, including, but not limited to parenterally, e.g. injectionsincluding, but not limited to, subcutaneously or intravenously or anyother form of injections or infusions. Polypeptide compositions can beadministered by a number of routes including, but not limited to oral,intravenous, intraperitoneal, intramuscular, transdermal, subcutaneous,topical, sublingual, or rectal means. Compositions comprisingnon-natural amino acid polypeptides, modified or unmodified, can also beadministered via liposomes. Such administration routes and appropriateformulations are generally known to those of skill in the art. The hGHpolypeptide comprising a non-natural amino acid, including PEGylatedhGH, may be used alone or in combination with other suitable componentssuch as a pharmaceutical carrier.

Average quantities of the hGH may vary and in particular should be basedupon the recommendations and prescription of a qualified physician. Theexact amount of hGH is a matter of preference subject to such factors asthe exact type of condition being treated, the condition of the patientbeing treated, as well as the other ingredients in the composition. Theamount to be given may be readily determined by one of ordinary skill inthe art based upon therapy with hGH.

Pharmaceutical compositions of the invention may be manufactured inconventional manner.

XI. General Recombinant Nucleic Acid Methods for Use with the Invention

In numerous embodiments of the present invention, nucleic acids encodinga hGH polypeptide of interest will be isolated, cloned and often alteredusing recombinant methods. Such embodiments are used, including but notlimited to, for protein expression or during the generation of variants,derivatives, expression cassettes, or other sequences derived from a hGHpolypeptide. In some embodiments, the sequences encoding thepolypeptides of the invention are operably linked to a heterologouspromoter. Isolation of hGH and production of GH in host cells aredescribed in, e.g., U.S. Pat. Nos. 4,601,980, 4,604,359, 4,634,677,4,658,021, 4,898,830, 5,424,199, 5,795,745, 5,854,026, 5,849,535;6,004,931; 6,022,711; 6,143,523 and 6,608,183, which are incorporated byreference herein.

A nucleotide sequence encoding a hGH polypeptide comprising anon-naturally encoded amino acid may be synthesized on the basis of theamino acid sequence of the parent polypeptide and then changing thenucleotide sequence so as to effect introduction (i.e., incorporation orsubstitution) or removal (i.e., deletion or substitution) of therelevant amino acid residue(s). The nucleotide sequence may beconveniently modified by site-directed mutagenesis in accordance withconventional methods. Alternatively, the nucleotide sequence may beprepared by chemical synthesis, including but not limited to, by usingan oligonucleotide synthesizer, wherein oligonucleotides are designedbased on the amino acid sequence of the desired polypeptide, andpreferably selecting those codons that are favored in the host cell inwhich the recombinant polypeptide will be produced. For example, severalsmall oligonucleotides coding for portions of the desired polypeptidemay be synthesized and assembled by PCR, ligation or ligation chainreaction. See, e.g., Barany, et al., Proc. Natl. Acad. Sci. 88: 189-193(1991); U.S. Pat. No. 6,521,427 which are incorporated by referenceherein.

This invention utilizes routine techniques in the field of recombinantgenetics. Basic texts disclosing the general methods of use in thisinvention include Sambrook et al., Molecular Cloning, A LaboratoryManual (3rd ed. 2001); Kriegler, Gene Transfer and Expression: ALaboratory Manual (1990); and Current Protocols in Molecular Biology(Ausubel et al., eds., 1994)).

General texts which describe molecular biological techniques includeBerger and Kimmel, Guide to Molecular Cloning Techniques Methods inEnzymology volume 152 Academic Press, Inc., San Diego, Calif. (Berger);Sambrook et al., Molecular Cloning—A Laboratory Manual (2nd Ed.), Vol.1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989(“Sambrook”) and Current Protocols in Molecular Biology, F. M. Ausubelet al., eds., Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc., (supplementedthrough 1999) (“Ausubel”)). These texts describe mutagenesis, the use ofvectors, promoters and many other relevant topics related to, includingbut not limited to, the generation of genes or polynucleotides thatinclude selector codons for production of proteins that includeunnatural amino acids, orthogonal tRNAs, orthogonal tRNA synthetases,and pairs thereof.

Various types of mutagenesis are used in the invention for a variety ofpurposes, including but not limited to, to produce novel synthetases ortRNAs, to mutate tRNA molecules, to mutate polynucleotides encodingsynthetases, to produce libraries of tRNAs, to produce libraries ofsynthetases, to produce selector codons, to insert selector codons thatencode unnatural amino acids in a protein or polypeptide of interest.They include but are not limited to site-directed, random pointmutagenesis, homologous recombination, DNA shuffling or other recursivemutagenesis methods, chimeric construction, mutagenesis using uracilcontaining templates, oligonucleotide-directed mutagenesis,phosphorothioate-modified DNA mutagenesis, mutagenesis using gappedduplex DNA or the like, or any combination thereof. Additional suitablemethods include point mismatch repair, mutagenesis usingrepair-deficient host strains, restriction-selection andrestriction-purification, deletion mutagenesis, mutagenesis by totalgene synthesis, double-strand break repair, and the like. Mutagenesis,including but not limited to, involving chimeric constructs, are alsoincluded in the present invention. In one embodiment, mutagenesis can beguided by known information of the naturally occurring molecule oraltered or mutated naturally occurring molecule, including but notlimited to, sequence, sequence comparisons, physical properties,secondary, tertiary, or quaternary structure, crystal structure or thelike.

The texts and examples found herein describe these procedures.Additional information is found in the following publications andreferences cited within: Ling et al., Approaches to DNA mutagenesis: anoverview, Anal Biochem. 254(2): 157-178 (1997); Dale et al.,Oligonucleotide-directed random mutagenesis using the phosphorothioatemethod, Methods Mol. Biol. 57:369-374 (1996); Smith, In vitromutagenesis, Ann. Rev. Genet. 19:423-462 (1985); Botstein & Shortle,Strategies and applications of in vitro mutagenesis, Science229:1193-1201 (1985); Carter, Site-directed mutagenesis, Biochem. J.237:1-7 (1986); Kunkel, The efficiency of oligonucleotide directedmutagenesis, in Nucleic Acids & Molecular Biology (Eckstein, F. andLilley, D. M. J. eds., Springer Verlag, Berlin) (1987); Kunkel, Rapidand efficient site-specific mutagenesis without phenotypic selection,Proc. Natl. Acad. Sci. USA 82:488-492 (1985); Kunkel et al., Rapid andefficient site-specific mutagenesis without phenotypic selection,Methods in Enzymol. 154, 367-382 (1987); Bass et al., Mutant Trprepressors with new DNA-binding specificities, Science 242:240-245(1988); Zoller & Smith, Oligonucleotide-directed mutagenesis usingM13-derived vectors: an efficient and general procedure for theproduction of point mutations in any DNA fragment, Nucleic Acids Res.10:6487-6500 (1982); Zoller & Smith, Oligonucleotide-directedmutagenesis of DNA fragments cloned into M13 vectors, Methods inEnzymol. 100:468-500 (1983); Zoller & Smith, Oligonucleotide-directedmutagenesis: a simple method using two oligonucleotide primers and asingle-stranded DNA template, Methods in Enzymol. 154:329-350 (1987);Taylor et al., The use of phosphorothioate-modified DNA in restrictionenzyme reactions to prepare nicked DNA, Nucl. Acids Res. 13: 8749-8764(1985); Taylor et al., The rapid generation of oligonucleotide-directedmutations at high frequency using phosphorothioate-modified DNA, Nucl.Acids Res. 13: 8765-8785 (1985); Nakamaye & Eckstein, Inhibition ofrestriction endonuclease Nci I cleavage by phosphorothioate groups andits application to oligonucleotide-directed mutagenesis, Nucl. AcidsRes. 14: 9679-9698 (1986); Sayers et al., 5′-3′ Exonucleases inphosphorothioate-based oligonucleotide-directed mutagenesis, Nucl. AcidsRes. 16:791-802 (1988); Sayers et al., Strand specific cleavage ofphosphorothioate-containing DNA by reaction with restrictionendonucleases in the presence of ethidium bromide, (1988) Nucl. AcidsRes. 16: 803-814; Kramer et al., The gapped duplex DNA approach tooligonucleotide-directed mutation construction, Nucl. Acids Res. 12:9441-9456 (1984); Kramer & Fritz Oligonucleotide-directed constructionof mutations via gapped duplex DNA, Methods in Enzymol. 154:350-367(1987); Kramer et al., Improved enzymatic in vitro reactions in thegapped duplex DNA approach to oligonucleotide-directed construction ofmutations, Nucl. Acids Res. 16: 7207 (1988); Fritz et al.,Oligonucleotide-directed construction of mutations: a gapped duplex DNAprocedure without enzymatic reactions in vitro, Nucl. Acids Res. 16:6987-6999 (1988); Kramer et al., Different base/base mismatches arecorrected with different efficiencies by the methyl-directed DNAmismatch-repair system of E. coli, Cell 38:879-887 (1984); Carter etal., Improved oligonucleotide site-directed mutagenesis using M13vectors, Nucl. Acids Res. 13: 4431-4443 (1985); Carter, Improvedoligonucleotide-directed mutagenesis using M13 vectors, Methods inEnzymol. 154: 382-403 (1987); Eghtedarzadeh & Henikoff, Use ofoligonucleotides to generate large deletions, Nucl. Acids Res. 14: 5115(1986); Wells et al., Importance of hydrogen-bond formation instabilizing the transition state of subtilisin, Phil. Trans. R. Soc.Lond. A 317: 415-423 (1986); Nambiar et al., Total synthesis and cloningof a gene coding for the ribonuclease S protein, Science 223: 1299-1301(1984); Sakmar and Khorana, Total synthesis and expression of a gene forthe alpha-subunit of bovine rod outer segment guanine nucleotide-bindingprotein (transducin), Nucl. Acids Res. 14: 6361-6372 (1988); Wells etal., Cassette mutagenesis: an efficient method for generation ofmultiple mutations at defined sites, Gene 34:315-323 (1985); Grundstromet al., Oligonucleotide-directed mutagenesis by microscale ‘shot-gun’gene synthesis, Nucl. Acids Res. 13: 3305-3316 (1985); Mandecki,Oligonucleotide-directed double-strand break repair in plasmids ofEscherichia coli: a method for site-specific mutagenesis, Proc. Natl.Acad. Sci. USA, 83:7177-7181 (1986); Arnold, Protein engineering forunusual environments, Current Opinion in Biotechnology 4:450-455 (1993);Sieber, et al., Nature Biotechnology, 19:456-460 (2001); W. P. C.Stemmer, Nature 370, 389-91 (1994); and, I. A. Lorimer, I. Pastan,Nucleic Acids Res. 23, 3067-8 (1995). Additional details on many of theabove methods can be found in Methods in Enzymology Volume 154, whichalso describes useful controls for trouble-shooting problems withvarious mutagenesis methods.

Oligonucleotides, e.g., for use in mutagenesis of the present invention,e.g., mutating libraries of synthetases, or altering tRNAs, aretypically synthesized chemically according to the solid phasephosphoramidite triester method described by Beaucage and Caruthers,Tetrahedron Letts. 22(20):1859-1862, (1981) e.g., using an automatedsynthesizer, as described in Needham-VanDevanter et al., Nucleic AcidsRes., 12:6159-6168 (1984).

The invention also relates to eukaryotic host cells, non-eukaryotic hostcells, and organisms for the in vivo incorporation of an unnatural aminoacid via orthogonal tRNA/RS pairs. Host cells are genetically engineered(including but not limited to, transformed, transduced or transfected)with the polynucleotides of the invention or constructs which include apolynucleotide of the invention, including but not limited to, a vectorof the invention, which can be, for example, a cloning vector or anexpression vector. For example, the coding regions for the orthogonaltRNA, the orthogonal tRNA synthetase, and the protein to be derivatizedare operably linked to gene expression control elements that arefunctional in the desired host cell. The vector can be, for example, inthe form of a plasmid, a cosmid, a phage, a bacterium, a virus, a nakedpolynucleotide, or a conjugated polynucleotide. The vectors areintroduced into cells and/or microorganisms by standard methodsincluding electroporation (Fromm et al., Proc. Natl. Acad. Sci. USA 82,5824 (1985)), infection by viral vectors, high velocity ballisticpenetration by small particles with the nucleic acid either within thematrix of small beads or particles, or on the surface (Klein et al.,Nature 327, 70-73 (1987)), and/or the like.

The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for such activities as, for example, screeningsteps, activating promoters or selecting transformants. These cells canoptionally be cultured into transgenic organisms. Other usefulreferences, including but not limited to for cell isolation and culture(e.g., for subsequent nucleic acid isolation) include Freshney (1994)Culture of Animal Cells, a Manual of Basic Technique, third edition,Wiley-Liss, New York and the references cited therein; Payne et al.(1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley &Sons, Inc. New York, N.Y.; Gamborg and Phillips (eds.) (1995) PlantCell, Tissue and Organ Culture; Fundamental Methods Springer Lab Manual,Springer-Verlag (Berlin Heidelberg N.Y.) and Atlas and Parks (eds.) TheHandbook of Microbiological Media (1993) CRC Press, Boca Raton, Fla.

Several well-known methods of introducing target nucleic acids intocells are available, any of which can be used in the invention. Theseinclude: fusion of the recipient cells with bacterial protoplastscontaining the DNA, electroporation, projectile bombardment, andinfection with viral vectors (discussed further, below), etc. Bacterialcells can be used to amplify the number of plasmids containing DNAconstructs of this invention. The bacteria are grown to log phase andthe plasmids within the bacteria can be isolated by a variety of methodsknown in the art (see, for instance, Sambrook). In addition, kits arecommercially available for the purification of plasmids from bacteria,(see, e.g., EASYPREP®, FLEXIPREP®, both from Pharmacia Biotech;STRATACLEANT™ from Stratagene; and, QIAPREP™ from Qiagen). The isolatedand purified plasmids are then further manipulated to produce otherplasmids, used to transfect cells or incorporated into related vectorsto infect organisms. Typical vectors contain transcription andtranslation terminators, transcription and translation initiationsequences, and promoters useful for regulation of the expression of theparticular target nucleic acid. The vectors optionally comprise genericexpression cassettes containing at least one independent terminatorsequence, sequences permitting replication of the cassette ineukaryotes, or prokaryotes, or both, (including but not limited to,shuttle vectors) and selection markers for both prokaryotic andeukaryotic systems. Vectors are suitable for replication and integrationin prokaryotes, eukaryotes, or both. See, Gillam & Smith, Gene 8:81(1979); Roberts, et al., Nature, 328:731 (1987); Schneider, E., et al.,Protein Expr. Purif. 6(1):10-14 (1995); Ausubel, Sambrook, Berger (allsupra). A catalogue of bacteria and bacteriophages useful for cloning isprovided, e.g., by the ATCC, e.g., The ATCC Catalogue of Bacteria andBacteriophage (1992) Gherna et al. (eds) published by the ATCC.Additional basic procedures for sequencing, cloning and other aspects ofmolecular biology and underlying theoretical considerations are alsofound in Watson et al. (1992) Recombinant DNA Second Edition ScientificAmerican Books, N.Y. In addition, essentially any nucleic acid (andvirtually any labeled nucleic acid, whether standard or non-standard)can be custom or standard ordered from any of a variety of commercialsources, such as the Midland Certified Reagent Company (Midland, Tex.available on the World Wide Web at mcrc.com), The Great American GeneCompany (Ramona, Calif. available on the World Wide Web at genco.com),ExpressGen Inc. (Chicago, Ill. available on the World Wide Web atexpressgen.com), Operon Technologies Inc. (Alameda, Calif.) and manyothers.

XII. Expression in Non-Eukaryotes and Eukaryotes

To obtain high level expression of a cloned hGH polynucleotide, onetypically subclones polynucleotides encoding a hGH polypeptide of theinvention into an expression vector that contains a strong promoter todirect transcription, a transcription/translation terminator, and if fora nucleic acid encoding a protein, a ribosome binding site fortranslational initiation. Suitable bacterial promoters are known tothose of ordinary skill in the art and described, e.g., in Sambrook etal. and Ausubel et al.

Bacterial expression systems for expressing hGH polypeptides of theinvention are available in, including but not limited to, E. coli,Bacillus sp., Pseudomonas fluorescens, Pseudomonas aeruginosa,Pseudomonas putida, and Salmonella (Palva et al., Gene 22:229-235(1983); Mosbach et al., Nature 302:543-545 (1983)). Kits for suchexpression systems are commercially available. Eukaryotic expressionsystems for mammalian cells, yeast, and insect cells are known to thoseof ordinary skill in the art and are also commercially available. Incases where orthogonal tRNAs and aminoacyl tRNA synthetases are used toexpress the hGH polypeptides of the invention, host cells for expressionare selected based on their ability to use the orthogonal components.Exemplary host cells include Gram-positive bacteria (including but notlimited to B. brevis, B. subtilis, or Streptomyces) and Gram-negativebacteria (E. coli, Pseudomonas fluorescens, Pseudomonas aeruginosa,Pseudomonas putida), as well as yeast and other eukaryotic cells. Cellscomprising O-tRNA/O-RS pairs can be used as described herein.

A eukaryotic host cell or non-eukaryotic host cell of the presentinvention provides the ability to synthesize proteins that compriseunnatural amino acids in large useful quantities. In one aspect, thecomposition optionally includes, including but not limited to, at least10 micrograms, at least 50 micrograms, at least 75 micrograms, at least100 micrograms, at least 200 micrograms, at least 250 micrograms, atleast 500 micrograms, at least 1 milligram, at least 10 milligrams, atleast 100 milligrams, at least one gram, or more of the protein thatcomprises an unnatural amino acid, or an amount that can be achievedwith in vivo protein production methods (details on recombinant proteinproduction and purification are provided herein). In another aspect, theprotein is optionally present in the composition at a concentration of,including but not limited to, at least 10 micrograms of protein perliter, at least 50 micrograms of protein per liter, at least 75micrograms of protein per liter, at least 100 micrograms of protein perliter, at least 200 micrograms of protein per liter, at least 250micrograms of protein per liter, at least 500 micrograms of protein perliter, at least 1 milligram of protein per liter, or at least 10milligrams of protein per liter or more, in, including but not limitedto, a cell lysate, a buffer, a pharmaceutical buffer, or other liquidsuspension (including but not limited to, in a volume of, including butnot limited to, anywhere from about 1 nl to about 100 L or more). Theproduction of large quantities (including but not limited to, greaterthat that typically possible with other methods, including but notlimited to, in vitro translation) of a protein in a eukaryotic cell ornon-eukaryotic cell including at least one unnatural amino acid is afeature of the invention.

A eukaryotic host cell or non-eukaryotic host cell of the presentinvention provides the ability to biosynthesize proteins that compriseunnatural amino acids in large useful quantities. For example, proteinscomprising an unnatural amino acid can be produced at a concentrationof, including but not limited to, at least 10 μg/liter, at least 50μg/liter, at least 75 μg/liter, at least 100 μg/liter, at least 200μg/liter, at least 250 μg/liter, or at least 500 μg/liter, at least 1mg/liter, at least 2 mg/liter, at least 3 mg/liter, at least 4 mg/liter,at least 5 mg/liter, at least 6 mg/liter, at least 7 mg/liter, at least8 mg/liter, at least 9 mg/liter, at least 10 mg/liter, at least 20, 30,40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900mg/liter, 1 g/liter, 5 g/liter, 10 g/liter or more of protein in a cellextract, cell lysate, culture medium, a buffer, and/or the like.

Expression Systems, Culture, and Isolation

hGH may be expressed in any number of suitable expression systemsincluding, for example, yeast, insect cells, mammalian cells, andbacteria. A description of exemplary expression systems is providedbelow.

Yeast

As used herein, the term “yeast” includes any of the various yeastscapable of expressing a gene encoding hGH. Such yeasts include, but arenot limited to, ascosporogenous yeasts (Endomycetales),basidiosporogenous yeasts and yeasts belonging to the Fungi imperfecti(Blastomycetes) group. The ascosporogenous yeasts are divided into twofamilies, Spermophthoraceae and Saccharomycetaceae. The latter iscomprised of four subfamilies, Schizosaccharomycoideae (e.g., genusSchizosaccharomyces), Nadsonioideae, Lipomycoideae and Saccharomycoideae(e.g., genera Pichia, Kluyveromyces and Saccharomyces). Thebasidiosporogenous yeasts include the genera Leucosporidium,Rhodosporidium, Sporidiobolus, Filobasidium and Filobasidiella. Yeastsbelonging to the Fungi Imperfecti (Blastomycetes) group are divided intotwo families, Sporobolomycetaceae (e.g., genera Sporobolomyces andBullera) and Cryptococcaceae (e.g., genus Candida).

Of particular interest for use with the present invention are specieswithin the genera Pichia, Kluyveromyces, Saccharomyces,Schizosaccharomyces, Hansenula, Torulopsis and Candida, including, butnot limited to, P. pastoris, P. guillerimondii, S. cerevisiae, S.carlsbergensis, S. diastaticus, S. douglasii, S. kluyveri, S. norbensis,S. oviformis, K. lactis, K. fragilis, C. albicans, C. maltosa, and H.polymorpha.

The selection of suitable yeast for expression of hGH is within theskill of one of ordinary skill in the art. In selecting yeast hosts forexpression, suitable hosts may include those shown to have, for example,good secretion capacity, low proteolytic activity, and overallrobustness. Yeast are generally available from a variety of sourcesincluding, but not limited to, the Yeast Genetic Stock Center,Department of Biophysics and Medical Physics, University of California(Berkeley, Calif.), and the American Type Culture Collection (“ATCC”)(Manassas, Va.).

The term “yeast host” or “yeast host cell” includes yeast that can be,or has been, used as a recipient for recombinant vectors or othertransfer DNA. The term includes the progeny of the original yeast hostcell that has received the recombinant vectors or other transfer DNA. Itis understood that the progeny of a single parental cell may notnecessarily be completely identical in morphology or in genomic or totalDNA complement to the original parent, due to accidental or deliberatemutation. Progeny of the parental cell that are sufficiently similar tothe parent to be characterized by the relevant property, such as thepresence of a nucleotide sequence encoding an hGH, are included in theprogeny intended by this definition.

Expression and transformation vectors, including extrachromosomalreplicons or integrating vectors, have been developed for transformationinto many yeast hosts. For example, expression vectors have beendeveloped for S. cerevisiae (Sikorski et al., GENETICS (1989) 122:19;Ito et al., J. BACTERIOL. (1983) 153:163; Hinnen et al., PROC. NATL.ACAD. S_(CI.) USA (1978) 75:1929); C. albicans (Kurtz et al., MOL. CELL.BIOL. (1986) 6:142); C. maltosa (Kunze et al., J. BASIC MICROBIOL.(1985) 25:141); H. polymorpha (Gleeson et al., J. GEN. MICROBIOL. (1986)132:3459; Roggenkamp et al., MOL. GENETICS AND GENOMICS (1986) 202:302);K. fragilis (Das et al., J. BACTERIOL. (1984) 158:1165); K. lactis (DeLouvencourt et al., J. BACTERIOL. (1983) 154:737; Van den Berg et al.,BIOTECHNOLOGY (NY) (1990) 8:135); P. guillerimondii (Kunze et al., J.BASIC MICROBIOL. (1985) 25:141); P. pastoris (U.S. Pat. Nos. 5,324,639;4,929,555; and 4,837,148; Cregg et al., MOL. CELL. BIOL. (1985) 5:3376);Schizosaccharomyces pombe (Beach et al., NATURE (1982) 300:706); and Y.lipolytica; A. nidulans (Ballance et al., BIOCHEM. BIOPHYS. RES. COMMUN.(1983) 112:284-89; Tilburn et al., GENE (1983) 26:205-221; and Yelton etal., PROC. NATL. ACAD. SCI. USA (1984) 81:1470-74); A. niger (Kelly andHynes, EMBO J. (1985) 4:475-479); T. reesia (EP 0 244 234); andfilamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium(WO 91/00357), each of which is incorporated by reference herein.

Control sequences for yeast vectors are known to those of ordinary skillin the art and include, but are not limited to, promoter regions fromgenes such as alcohol dehydrogenase (ADH) (EP 0 284 044); enolase;glucokinase; glucose-6-phosphate isomerase;glyceraldehyde-3-phosphate-dehydrogenase (GAP or GAPDH); hexokinase;phosphofructokinase; 3-phosphoglycerate mutase; and pyruvate kinase(PyK) (EP 0 329 203). The yeast PHO5 gene, encoding-acid phosphatase,also may provide useful promoter sequences (Miyanohara et al., PROC.NATL. ACAD. SCI. USA (1983) 80:1). Other suitable promoter sequences foruse with yeast hosts may include the promoters for 3-phosphoglyceratekinase (Hitzeman et al., J. BIOL. CHEM. (1980) 255:12073; and otherglycolytic enzymes, such as pyruvate decarboxylase, triosephosphateisomerase, and phosphoglucose isomerase (Holland et al., BIOCHEMISTRY(1978) 17:4900; Hess et al., J. ADV. ENZYME REG. (1969) 7:149).Inducible yeast promoters having the additional advantage oftranscription controlled by growth conditions may include the promoterregions for alcohol dehydrogenase 2; isocytochrome C; acid phosphatase;metallothionein; glyceraldehyde-3-phosphate dehydrogenase; degradativeenzymes associated with nitrogen metabolism; and enzymes responsible formaltose and galactose utilization. Suitable vectors and promoters foruse in yeast expression are further described in EP 0 073 657.

Yeast enhancers also may be used with yeast promoters. In addition,synthetic promoters may also function as yeast promoters. For example,the upstream activating sequences (UAS) of a yeast promoter may bejoined with the transcription activation region of another yeastpromoter, creating a synthetic hybrid promoter. Examples of such hybridpromoters include the ADH regulatory sequence linked to the GAPtranscription activation region. See U.S. Pat. Nos. 4,880,734 and4,876,197. Other examples of hybrid promoters include promoters thatconsist of the regulatory sequences of the ADH2, GAL4, GAL10, or PHO5genes, combined with the transcriptional activation region of aglycolytic enzyme gene such as GAP or PyK. See EP 0 164 556.Furthermore, a yeast promoter may include naturally occurring promotersof non-yeast origin that have the ability to bind yeast RNA polymeraseand initiate transcription.

Other control elements that may comprise part of the yeast expressionvectors include terminators, for example, from GAPDH or the enolasegenes (Holland et al., J. BIOL. CHEM. (1981) 256:1385). In addition, theorigin of replication from the 2μ plasmid origin is suitable for yeast.A suitable selection gene for use in yeast is the trp1 gene present inthe yeast plasmid. See Tschumper et al., GENE (1980) 10:157; Kingsman etal., GENE (1979) 7:141. The trp1 gene provides a selection marker for amutant strain of yeast lacking the ability to grow in tryptophan.Similarly, Leu2-deficient yeast strains (ATCC 20,622 or 38,626) arecomplemented by known plasmids bearing the Leu2 gene.

Methods of introducing exogenous DNA into yeast hosts are known to thoseof ordinary skill in the art, and typically include, but are not limitedto, either the transformation of spheroplasts or of intact yeast hostcells treated with alkali cations. For example, transformation of yeastcan be carried out according to the method described in Hsiao et al.,PROC. NATL. ACAD. SCI. USA (1979) 76:3829 and Van Solingen et al., J.BACT. (1977) 130:946. However, other methods for introducing DNA intocells such as by nuclear injection, electroporation, or protoplastfusion may also be used as described generally in SAMBROOK ET AL.,MOLECULAR CLONING: A LAB. MANUAL (2001). Yeast host cells may then becultured using standard techniques known to those of ordinary skill inthe art.

Other methods for expressing heterologous proteins in yeast host cellsare known to those of ordinary skill in the art. See generally U.S.Patent Application No. 20020055169, U.S. Pat. Nos. 6,361,969; 6,312,923;6,183,985; 6,083,723; 6,017,731; 5,674,706; 5,629,203; 5,602,034; and5,089,398; U.S. Reexamined Pat. Nos. RE37,343 and RE35,749; PCTPublished Patent Applications WO 99/078621; WO 98/37208; and WO98/26080; European Patent Applications EP 0 946 736; EP 0 732 403; EP 0480 480; WO 90/10277; EP 0 340 986; EP 0 329 203; EP 0 324 274; and EP 0164 556, which are incorporated by reference herein. See also Gellissenet al., ANTONIE VAN LEEUWENHOEK (1992) 62(1-2):79-93; Romanos et al.,YEAST (1992) 8(6):423-488; Goeddel, METHODS IN ENZYMOLOGY (1990)185:3-7, each is incorporated by reference herein.

The yeast host strains may be grown in fermentors during theamplification stage using standard feed batch fermentation methods knownto those of ordinary skill in the art. The fermentation methods may beadapted to account for differences in a particular yeast host's carbonutilization pathway or mode of expression control. For example,fermentation of a Saccharomyces yeast host may require a single glucosefeed, complex nitrogen source (e.g., casein hydrolysates), and multiplevitamin supplementation. In contrast, the methylotrophic yeast P.pastoris may require glycerol, methanol, and trace mineral feeds, butonly simple ammonium (nitrogen) salts for optimal growth and expression.See, e.g., U.S. Pat. No. 5,324,639; Elliott et al., J. PROTEIN CHEM.(1990) 9:95; and Fieschko et al., BIOTECH. BIOENG. (1987) 29:1113.

Such fermentation methods, however, may have certain common featuresindependent of the yeast host strain employed. For example, a growthlimiting nutrient, typically carbon, may be added to the fermentorduring the amplification phase to allow maximal growth. In addition,fermentation methods generally employ a fermentation medium designed tocontain adequate amounts of carbon, nitrogen, basal salts, phosphorus,and other minor nutrients (vitamins, trace minerals and salts, etc.).Examples of fermentation media suitable for use with Pichia aredescribed in U.S. Pat. Nos. 5,324,639 and 5,231,178, which areincorporated by reference herein.

Baculovirus-Infected Insect Cells

The term “insect host” or “insect host cell” refers to a insect that canbe, or has been, used as a recipient for recombinant vectors or othertransfer DNA. The term includes the progeny of the original insect hostcell that has been transfected. It is understood that the progeny of asingle parental cell may not necessarily be completely identical inmorphology or in genomic or total DNA complement to the original parent,due to accidental or deliberate mutation. Progeny of the parental cellthat are sufficiently similar to the parent to be characterized by therelevant property, such as the presence of a nucleotide sequenceencoding an hGH polypeptide, are included in the progeny intended bythis definition.

The selection of suitable insect cells for expression of hGH is known tothose of ordinary skill in the art. Several insect species are welldescribed in the art and are commercially available including Aedesaegypti, Bombyx mori, Drosophila melanogaster, Spodoptera frugiperda,and Trichoplusia ni. In selecting insect hosts for expression, suitablehosts may include those shown to have, inter alia, good secretioncapacity, low proteolytic activity, and overall robustness. Insect aregenerally available from a variety of sources including, but not limitedto, the Insect Genetic Stock Center, Department of Biophysics andMedical Physics, University of California (Berkeley, Calif.); and theAmerican Type Culture Collection (“ATCC”) (Manassas, Va.).

Generally, the components of a baculovirus-infected insect expressionsystem include a transfer vector, usually a bacterial plasmid, whichcontains both a fragment of the baculovirus genome, and a convenientrestriction site for insertion of the heterologous gene to be expressed;a wild type baculovirus with a sequences homologous to thebaculovirus-specific fragment in the transfer vector (this allows forthe homologous recombination of the heterologous gene in to thebaculovirus genome); and appropriate insect host cells and growth media.The materials, methods and techniques used in constructing vectors,transfecting cells, picking plaques, growing cells in culture, and thelike are known in the art and manuals are available describing thesetechniques.

After inserting the heterologous gene into the transfer vector, thevector and the wild type viral genome are transfected into an insecthost cell where the vector and viral genome recombine. The packagedrecombinant virus is expressed and recombinant plaques are identifiedand purified. Materials and methods for baculovirus/insect cellexpression systems are commercially available in kit form from, forexample, Invitrogen Corp. (Carlsbad, Calif.). These techniques aregenerally known to those of ordinary skill in the art and fullydescribed in SUMMERS AND SMITH, TEXAS AGRICULTURAL EXPERIMENT STATIONBULLETIN NO. 1555 (1987), herein incorporated by reference. See also,RICHARDSON, 39 METHODS IN MOLECULAR BIOLOGY: BACULOVIRUS EXPRESSIONPROTOCOLS (1995); AUSUBEL ET AL., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY16.9-16.11 (1994); KING AND POSSEE, THE BACULOVIRUS SYSTEM: A LABORATORYGUIDE (1992); and O'REILLY ET AL., BACULOVIRUS EXPRESSION VECTORS: ALABORATORY MANUAL (1992).

Indeed, the production of various heterologous proteins usingbaculovirus/insect cell expression systems is known to those of ordinaryskill in the art. See, e.g., U.S. Pat. Nos. 6,368,825; 6,342,216;6,338,846; 6,261,805; 6,245,528, 6,225,060; 6,183,987; 6,168,932;6,126,944; 6,096,304; 6,013,433; 5,965,393; 5,939,285; 5,891,676;5,871,986; 5,861,279; 5,858,368; 5,843,733; 5,762,939; 5,753,220;5,605,827; 5,583,023; 5,571,709; 5,516,657; 5,290,686; WO 02/06305; WO01/90390; WO 01/27301; WO 01/05956; WO 00/55345; WO 00/20032; WO99/51721; WO 99/45130; WO 99/31257; WO 99/10515; WO 99/09193; WO97/26332; WO 96/29400; WO 96/25496; WO 96/06161; WO 95/20672; WO93/03173; WO 92/16619; WO 92/02628; WO 92/01801; WO 90/14428; WO90/10078; WO 90/02566; WO 90/02186; WO 90/01556; WO 89/01038; WO89/01037; WO 88/07082, which are incorporated by reference herein.

Vectors that are useful in baculovirus/insect cell expression systemsare known in the art and include, for example, insect expression andtransfer vectors derived from the baculovirus Autographa californicanuclear polyhedrosis virus (AcNPV), which is a helper-independent, viralexpression vector. Viral expression vectors derived from this systemusually use the strong viral polyhedrin gene promoter to driveexpression of heterologous genes. See generally, O'Reilly ET AL.,BACULOVIRUS EXPRESSION VECTORS: A LABORATORY MANUAL (1992).

Prior to inserting the foreign gene into the baculovirus genome, theabove-described components, comprising a promoter, leader (if desired),coding sequence of interest, and transcription termination sequence, aretypically assembled into an intermediate transplacement construct(transfer vector). Intermediate transplacement constructs are oftenmaintained in a replicon, such as an extra chromosomal element (e.g.,plasmids) capable of stable maintenance in a host, such as bacteria. Thereplicon will have a replication system, thus allowing it to bemaintained in a suitable host for cloning and amplification. Morespecifically, the plasmid may contain the polyhedrin polyadenylationsignal (Miller, ANN. REV. MICROBIOL. (1988) 42:177) and a prokaryoticampicillin-resistance (amp) gene and origin of replication for selectionand propagation in E. coli.

One commonly used transfer vector for introducing foreign genes intoAcNPV is pAc373. Many other vectors, known to those of skill in the art,have also been designed including, for example, pVL985, which alters thepolyhedrin start codon from ATG to ATT, and which introduces a BamHIcloning site 32 base pairs downstream from the ATT. See Luckow andSummers, VIROLOGY 170:31 (1989). Other commercially available vectorsinclude, for example, PBlueBac4.5/V5-His; pBlueBacHis2; pMelBac;pBlueBac4.5 (Invitrogen Corp., Carlsbad, Calif.).

After insertion of the heterologous gene, the transfer vector and wildtype baculoviral genome are co-transfected into an insect cell host.Methods for introducing heterologous DNA into the desired site in thebaculovirus virus are known in the art. See SUMMERS AND SMITH, TEXASAGRICULTURAL EXPERIMENT STATION BULLETIN NO. 1555 (1987); Smith et al.,MOL. CELL. BIOL. (1983) 3:2156; Luckow and Summers, VIROLOGY (1989)170:31. For example, the insertion can be into a gene such as thepolyhedrin gene, by homologous double crossover recombination; insertioncan also be into a restriction enzyme site engineered into the desiredbaculovirus gene. See Miller et al., BIOESSAYS (1989) 11(4):91.

Transfection may be accomplished by electroporation. See TROTTER ANDWOOD, 39 METHODS IN MOLECULAR BIOLOGY (1995); Mann and King, J. GEN.VIROL. (1989) 70:3501. Alternatively, liposomes may be used to transfectthe insect cells with the recombinant expression vector and thebaculovirus. See, e.g., Liebman et al., BIOTECHNIQUES (1999) 26(1):36;Graves et al., BIOCHEMISTRY (1998) 37:6050; Nomura et al., J. BIOL.CHEM. (1998) 273(22):13570; Schmidt et al., PROTEIN EXPRESSION ANDPURIFICATION (1998) 12:323; Siffert et al., NATURE GENETICS (1998)18:45; TILKINS ET AL., CELL BIOLOGY: A LABORATORY HANDBOOK 145-154(1998); Cai et al., PROTEIN EXPRESSION AND PURIFICATION (1997) 10:263;Dolphin et al., NATURE GENETICS (1997) 17:491; Kost et al., GENE (1997)190:139; Jakobsson et al., J. BIOL. CHEM. (1996) 271:22203; Rowles etal., J. BIOL. CHEM. (1996) 271(37):22376; Reverey et al., J. BIOL. CHEM.(1996) 271(39):23607-10; Stanley et al., J. BIOL. CHEM. (1995) 270:4121;Sisk et al., J. VIROL. (1994) 68(2):766; and Peng et al., BIOTECHNIQUES(1993) 14(2):274. Commercially available liposomes include, for example,Cellfectin® and Lipofectin® (Invitrogen, Corp., Carlsbad, Calif.). Inaddition, calcium phosphate transfection may be used. See TROTTER ANDWOOD, 39 METHODS IN MOLECULAR BIOLOGY (1995); Kitts, NAR (1990)18(19):5667; and Mann and King, J. GEN. VIROL. (1989) 70:3501.

Baculovirus expression vectors usually contain a baculovirus promoter. Abaculovirus promoter is any DNA sequence capable of binding abaculovirus RNA polymerase and initiating the downstream (3′)transcription of a coding sequence (e.g., structural gene) into mRNA. Apromoter will have a transcription initiation region which is usuallyplaced proximal to the 5′ end of the coding sequence. This transcriptioninitiation region typically includes an RNA polymerase binding site anda transcription initiation site. A baculovirus promoter may also have asecond domain called an enhancer, which, if present, is usually distalto the structural gene. Moreover, expression may be either regulated orconstitutive.

Structural genes, abundantly transcribed at late times in the infectioncycle, provide particularly useful promoter sequences. Examples includesequences derived from the gene encoding the viral polyhedron protein(FRIESEN ET AL., The Regulation of Baculovirus Gene Expression in THEMOLECULAR BIOLOGY OF BACULOVIRUSES (1986); EP 0 127 839 and 0 155 476)and the gene encoding the p10 protein (Vlak et al., J. GEN. VIROL.(1988) 69:765.

The newly formed baculovirus expression vector is packaged into aninfectious recombinant baculovirus and subsequently grown plaques may bepurified by techniques known to those of ordinary skill in the art. SeeMiller et al., BIOESSAYS (1989) 11(4):91; SUMMERS AND SMITH, TEXASAGRICULTURAL EXPERIMENT STATION BULLETIN NO. 1555 (1987).

Recombinant baculovirus expression vectors have been developed forinfection into several insect cells. For example, recombinantbaculoviruses have been developed for, inter alia, Aedes aegypti (ATCCNo. CCL-125), Bombyx mori (ATCC No. CRL-8910), Drosophila melanogaster(ATCC No. 1963), Spodoptera frugiperda, and Trichoplusia ni. See Wright,NATURE (1986) 321:718; Carbonell et al., J. VIROL. (1985) 56:153; Smithet al., MOL. CELL. BIOL. (1983) 3:2156. See generally, Fraser et al., INVITRO CELL. DEV. BIOL. (1989) 25:225. More specifically, the cell linesused for baculovirus expression vector systems commonly include, but arenot limited to, Sf9 (Spodoptera frugiperda) (ATCC No. CRL-1711), Sf21(Spodoptera frugiperda) (Invitrogen Corp., Cat. No. 11497-013 (Carlsbad,Calif.)), Tri-368 (Trichopulsia ni), and HIGH-FIVE™ BTI-TN-5B1-4(Trichopulsia ni).

Cells and culture media are commercially available for both direct andfusion expression of heterologous polypeptides in abaculovirus/expression, and cell culture technology is generally knownto those of ordinary skill in the art.

E. coli, Pseudomonas species, and other Prokaryotes

Bacterial expression techniques are known to those of ordinary skill inthe art. A wide variety of vectors are available for use in bacterialhosts. The vectors may be single copy or low or high multicopy vectors.Vectors may serve for cloning and/or expression. In view of the ampleliterature concerning vectors, commercial availability of many vectors,and even manuals describing vectors and their restriction maps andcharacteristics, no extensive discussion is required here. As iswell-known, the vectors normally involve markers allowing for selection,which markers may provide for cytotoxic agent resistance, prototrophy orimmunity. Frequently, a plurality of markers are present, which providefor different characteristics.

A bacterial promoter is any DNA sequence capable of binding bacterialRNA polymerase and initiating the downstream (3′) transcription of acoding sequence (e.g. structural gene) into mRNA. A promoter will have atranscription initiation region which is usually placed proximal to the5′ end of the coding sequence. This transcription initiation regiontypically includes an RNA polymerase binding site and a transcriptioninitiation site. A bacterial promoter may also have a second domaincalled an operator, that may overlap an adjacent RNA polymerase bindingsite at which RNA synthesis begins. The operator permits negativeregulated (inducible) transcription, as a gene repressor protein maybind the operator and thereby inhibit transcription of a specific gene.Constitutive expression may occur in the absence of negative regulatoryelements, such as the operator. In addition, positive regulation may beachieved by a gene activator protein binding sequence, which, if presentis usually proximal (5′) to the RNA polymerase binding sequence. Anexample of a gene activator protein is the catabolite activator protein(CAP), which helps initiate transcription of the lac operon inEscherichia coli (E. coli) [Raibaud et al., ANNU. REV. GENET. (1984)18:173]. Regulated expression may therefore be either positive ornegative, thereby either enhancing or reducing transcription.

Sequences encoding metabolic pathway enzymes provide particularly usefulpromoter sequences. Examples include promoter sequences derived fromsugar metabolizing enzymes, such as galactose, lactose (lac) [Chang etal., NATURE (1977) 198:1056], and maltose. Additional examples includepromoter sequences derived from biosynthetic enzymes such as tryptophan(trp) [Goeddel et al., NUC. ACIDS RES. (1980) 8:4057, Yelverton et al.,NUCL. ACIDS RES. (1981) 9:731; U.S. Pat. No. 4,738,921; EPO Pub. Nos.036 776 and 121 775, which are incorporated by reference herein]. Theg-lactamase (bla) promoter system [Weissmann (1981) “The cloning ofinterferon and other mistakes.” In Interferon 3 (Ed. I. Gresser)],bacteriophage lambda PL [Shimatake et al., NATURE (1981) 292:128] and T5[U.S. Pat. No. 4,689,406] promoter systems also provide useful promotersequences. Preferred methods of the present invention utilize strongpromoters, such as the T7 promoter to induce hGH at high levels.Examples of such vectors are known those of ordinary skill in the artand include the pET29 series from Novagen, and the pPOP vectorsdescribed in WO99/05297. Such expression systems produce high levels ofhGH in the host without compromising host cell viability or growthparameters. pET19 (Novagen) is another vector known in the art.

In addition, synthetic promoters which do not occur in nature alsofunction as bacterial promoters. For example, transcription activationsequences of one bacterial or bacteriophage promoter may be joined withthe operon sequences of another bacterial or bacteriophage promoter,creating a synthetic hybrid promoter [U.S. Pat. No. 4,551,433, which isincorporated by reference herein]. For example, the tac promoter is ahybrid trp-lac promoter comprised of both trp promoter and lac operonsequences that is regulated by the lac repressor [Amann et al., GENE(1983) 25:167; de Boer et al., PROC. NATL. ACAD. SCI. (1983) 80:21].Furthermore, a bacterial promoter can include naturally occurringpromoters of non-bacterial origin that have the ability to bindbacterial RNA polymerase and initiate transcription. A naturallyoccurring promoter of non-bacterial origin can also be coupled with acompatible RNA polymerase to produce high levels of expression of somegenes in prokaryotes. The bacteriophase T7 RNA polymerase/promotersystem is an example of a coupled promoter system [Studier et al., J.MOL. BIOL. (1986) 189:113; Tabor et al., Proc Natl. Acad. Sci. (1985)82:1074]. In addition, a hybrid promoter can also be comprised of abacteriophage promoter and an E. coli operator region (EPO Pub. No. 267851).

In addition to a functioning promoter sequence, an efficient ribosomebinding site is also useful for the expression of foreign genes inprokaryotes. In E. coli, the ribosome binding site is called theShine-Dalgarno (SD) sequence and includes an initiation codon (ATG) anda sequence 3-9 nucleotides in length located 3-11 nucleotides upstreamof the initiation codon [Shine et al., NATURE (1975) 254:34]. The SDsequence is thought to promote binding of mRNA to the ribosome by thepairing of bases between the SD sequence and the 3′ and of E. coli 16SrRNA [Steitz et al. “Genetic signals and nucleotide sequences inmessenger RNA”, In Biological Regulation and Development: GeneExpression (Ed. R. F. Goldberger, 1979)]. To express eukaryotic genesand prokaryotic genes with weak ribosome-binding site [Sambrook et al.“Expression of cloned genes in Escherichia coli”, Molecular Cloning: ALaboratory Manual, 1989].

The term “bacterial host” or “bacterial host cell” refers to a bacterialthat can be, or has been, used as a recipient for recombinant vectors orother transfer DNA. The term includes the progeny of the originalbacterial host cell that has been transfected. It is understood that theprogeny of a single parental cell may not necessarily be completelyidentical in morphology or in genomic or total DNA complement to theoriginal parent, due to accidental or deliberate mutation. Progeny ofthe parental cell that are sufficiently similar to the parent to becharacterized by the relevant property, such as the presence of anucleotide sequence encoding an hGH, are included in the progenyintended by this definition.

The selection of suitable host bacteria for expression of hGH is knownto those of ordinary skill in the art. In selecting bacterial hosts forexpression, suitable hosts may include those shown to have, inter alia,good inclusion body formation capacity, low proteolytic activity, andoverall robustness. Bacterial hosts are generally available from avariety of sources including, but not limited to, the Bacterial GeneticStock Center, Department of Biophysics and Medical Physics, Universityof California (Berkeley, Calif.); and the American Type CultureCollection (“ATCC”) (Manassas, Va.). Industrial/pharmaceuticalfermentation generally use bacteria derived from K strains (e.g. W3110)or bacteria derived from B strains (e.g. BL21). These strains areparticularly useful because their growth parameters are extremely wellknown and robust. In addition, these strains are non-pathogenic, whichis commercially important for safety and environmental reasons. In oneembodiment, the E. coli host is a strain of DH10B, including but notlimited to DH10B(fis). Other examples of suitable E. coli hosts include,but are not limited to, strains of BL21, DH10B, or derivatives thereof.In another embodiment, the E. coli host is a strain of W3110.Recombinant host cell strains may be modified by genetic mutation tooptimize for desired characteristics. For example, host cell strains maybe genetically modified to modulate the expression of metabolicallyimportant genes, such as those involved in carbon source metabolism,amino acid metabolism, or protease production. Such genes may be mutatedto decrease, increase, knock-out, or knock-in expression in the desiredhost strain. Strain W3110, for example, may be modified to effect agenetic mutation in one or more genes involved in the metabolism ofarabinose including, but not limited to, the araB gene. Strains, forexample, may be modified to effect a genetic mutation or knock out othergenes. Methods to mutate or knock out genes are known to one of ordinaryskill in the art. Strains may also be mutated to modulate endogenousprotease activity to increase the production of full length hGH and/orto minimize the need for the addition of exogenous chemical inhibitorsto proteases. Other host cell strains include but are not limited to,BL21. In another embodiment of the methods of the present invention, theE. coli host is a protease minus strain including, but not limited to,OMP- and LON-. The host cell strain may be a species of Pseudomonas,including but not limited to, Pseudomonas fluorescens, Pseudomonasaeruginosa, and Pseudomonas putida. Pseudomonas fluorescens biovar 1,designated strain MB 101, is known to be useful for recombinantproduction and is available for therapeutic protein productionprocesses. Examples of a Pseudomonas expression system include thesystem available from The Dow Chemical Company as a host strain(Midland, Mich. available on the World Wide Web at dow.com). U.S. Pat.Nos. 4,755,465 and 4,859,600, which are incorporated by referenceherein, describe the use of Pseudomonas strains as a host cell for hGHproduction.

Once a recombinant host cell strain has been established (i.e., theexpression construct has been introduced into the host cell and hostcells with the proper expression construct are isolated), therecombinant host cell strain is cultured under conditions appropriatefor production of hGH. As will be apparent to one of skill in the art,the method of culture of the recombinant host cell strain will bedependent on the nature of the expression construct utilized and theidentity of the host cell. Recombinant host strains are normallycultured using methods that are known to those of ordinary skill in theart. Recombinant host cells are typically cultured in liquid mediumcontaining assimilatable sources of carbon, nitrogen, and inorganicsalts and, optionally, containing vitamins, amino acids, growth factors,and other proteinaceous culture supplements known to those of ordinaryskill in the art. Media or feed composition and/or nutrient requirementsfor optimal growth may differ for different recombinant host cellsand/or for smaller vs. larger scale preparations. Required trace metalsor vitamins, for example, may be altered as growth conditions changeand/or alternative host cells are used. To optimize production of hGHpolypeptide, conditions suitable for induction may be altered dependingon the recombinant host cell used, the expression construct, and/ormodifications such as mutations made to the host cell, including, butnot limited to, alterations in arabinose levels for induction. Arabinoselevels in the fermentation may be between about 0.0001% to about 0.1%,including but not limited to, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%,0.04%, 0.03%, 0.02%, 0.01%, 0.0095%, 0.009%, 0.0085%, 0.008%, 0.0075%,0.007%, 0.0065%, 0.006%, 0.0055%, 0.005%, 0.0045%, 0.004%, 0.0035%,0.003%, 0.0025%, 0.002%, 0.0015%, 0.001%, 0.00095%, 0.0009%, 0.00085%,0.0008%, 0.00075%, 0.0007%, 0.00065%, 0.0006%, 0.00055%, 0.0005%,0.00045%, 0.0004%, 0.00035%, 0.0003%, 0.00025%, 0.0002%, 0.00015%,0.0001%. In some embodiments, the arabinose levels are between 0.0005%and 0.05%. In some embodiments, the arabinose levels are between 0.001%to 0.02%. Alterations to provide a higher cell density for harvest mayalso be performed; steps including, but not limited to, the addition ofa second feed may be performed. Liquid media for culture of host cellsmay optionally contain antibiotics or antifungals to prevent the growthof undesirable microorganisms and/or compounds including, but notlimited to, antibiotics to select for host cells containing theexpression vector. Recombinant host cells may be cultured in batch orcontinuous formats, with either cell harvesting (in the case where thevariant hGH accumulates intracellularly) or harvesting of culturesupernatant in either batch or continuous formats. For production inprokaryotic host cells, batch culture and cell harvest are preferred.

Modulated suppression, continuous suppression, or induced suppressionmay be performed. It is readily apparent to those of skill in the artthat the non-naturally encoded amino acid may be added to the cellculture at a wide variety of different times during cell growth, or maybe present continuously during cell growth. The addition of one or morenon-naturally encoded amino acid for incorporation into hGH may occurbefore induction, at the time of induction, or after induction of hGHexpression by the host cells. In one embodiment, the non-naturallyencoded amino acid is added before induction of hGH expression. In oneembodiment, the non-naturally encoded amino acid is added approximatelyone hour before induction. In another embodiment, the non-naturallyencoded amino acids is present throughout cell growth.

Recombinant host cells expressing hGH, whether soluble, secreted, orinsoluble, may be grown in a wide variety of culture volumes. Theprocesses of the present invention are amenable to small laboratoryscale culture volumes as well as large scale commercial scale volumes.It is readily apparent to one of ordinary skill in the art that theprocesses of the present invention disclosed herein are scalable tolarger culture volumes. Large scale commercial culture volumes may be ofa wide range from, for example, one or more liters each, to hundreds ofliters, thousands of liters, 5000 liters, 10,000 liters, 20,000 liters,30,000 liters, 40,000 liters, 50,000 liters, up to 100,000 liters ormore. In producing large scale volumes, modification to some steps ofthe process may be necessary and are readily apparent to those ofordinary skill in art.

The hGH of the invention are normally purified after expression inrecombinant systems. hGH may be purified from host cells or culturemedium by a variety of methods known to the art. hGH produced inbacterial host cells may be poorly soluble or insoluble (in the form ofinclusion bodies). In the case of insoluble protein, the protein may becollected from host cell lysates by centrifugation and may further befollowed by homogenization of the cells. In the case of poorly solubleprotein, compounds including, but not limited to, polyethylene imine(PEI) may be added to induce the precipitation of partially solubleprotein. The precipitated protein may then be conveniently collected bycentrifugation. Recombinant host cells may be disrupted or homogenizedto release the inclusion bodies from within the cells using a variety ofmethods known to those of ordinary skill in the art. Host celldisruption or homogenization may be performed using well knowntechniques including, but not limited to, enzymatic cell disruption,sonication, dounce homogenization, or high pressure release disruption.In one embodiment of the method of the present invention, the highpressure release technique is used to disrupt the E. coli host cells torelease the inclusion bodies of hGH.

Insoluble or precipitated hGH may then be solubilized using any of anumber of suitable solubilization agents known to the art. hGH may besolubilized with urea or guanidine hydrochloride. The volume of thesolubilized hGH should be minimized so that large batches may beproduced using conveniently manageable batch sizes. This factor may besignificant in a large-scale commercial setting where the recombinanthost may be grown in batches that are thousands of liters in volume. Inaddition, when manufacturing hGH in a large-scale commercial setting, inparticular for human pharmaceutical uses, the avoidance of harshchemicals that can damage the machinery and container, or the proteinproduct itself, should be avoided, if possible. It has been shown in themethod of the present invention that the milder denaturing agent ureacan be used to solubilize the hGH inclusion bodies in place of theharsher denaturing agent guanidine hydrochloride. The use of ureasignificantly reduces the risk of damage to stainless steel equipmentutilized in the manufacturing and purification process of hGH whileefficiently solubilizing the hGH inclusion bodies.

In the case of soluble hGH protein, the hGH may be secreted into theperiplasmic space or into the culture medium. In addition, soluble hGHmay be present in the cytoplasm of the host cells. It may be desired toconcentrate soluble hGH prior to performing purification steps. Standardtechniques known to those of ordinary skill in the art may be used toconcentrate soluble hGH from, for example, cell lysates or culturemedium. In addition, standard techniques known to those of ordinaryskill in the art may be used to disrupt host cells and release solublehGH from the cytoplasm or periplasmic space of the host cells.

When hGH is produced as a fusion protein, the fusion sequence may beremoved. Removal of a fusion sequence may be accomplished by enzymaticor chemical cleavage. Enzymatic removal of fusion sequences may beaccomplished using methods known to those of ordinary skill in the art.The choice of enzyme for removal of the fusion sequence will bedetermined by the identity of the fusion, and the reaction conditionswill be specified by the choice of enzyme as will be apparent to one ofordinary skill in the art. Chemical cleavage may be accomplished usingreagents known to those of ordinary skill in the art, including but notlimited to, cyanogen bromide, TEV protease, and other reagents. Thecleaved hGH may be purified from the cleaved fusion sequence by methodsknown to those of ordinary skill in the art. Such methods will bedetermined by the identity and properties of the fusion sequence and thehGH, as will be apparent to one of ordinary skill in the art. Methodsfor purification may include, but are not limited to, size-exclusionchromatography, hydrophobic interaction chromatography, ion-exchangechromatography or dialysis or any combination thereof.

hGH may also be purified to remove DNA from the protein solution. DNAmay be removed by any suitable method known to the art, such asprecipitation or ion exchange chromatography, but may be removed byprecipitation with a nucleic acid precipitating agent, such as, but notlimited to, protamine sulfate. hGH may be separated from theprecipitated DNA using standard well known methods including, but notlimited to, centrifugation or filtration. Removal of host nucleic acidmolecules is an important factor in a setting where the hGH is to beused to treat humans and the methods of the present invention reducehost cell DNA to pharmaceutically acceptable levels.

Methods for small-scale or large-scale fermentation can also be used inprotein expression, including but not limited to, fermentors, shakeflasks, fluidized bed bioreactors, hollow fiber bioreactors, rollerbottle culture systems, and stirred tank bioreactor systems. Each ofthese methods can be performed in a batch, fed-batch, or continuous modeprocess.

Human hGH polypeptides of the invention can generally be recovered usingmethods standard in the art. For example, culture medium or cell lysatecan be centrifuged or filtered to remove cellular debris. Thesupernatant may be concentrated or diluted to a desired volume ordiafiltered into a suitable buffer to condition the preparation forfurther purification. Purification of the hGH polypeptide may includeseparating deamidated and clipped forms of the hGH polypeptide variantfrom the intact form.

Any of the following exemplary procedures can be employed forpurification of hGH polypeptides of the invention: affinitychromatography; anion- or cation-exchange chromatography (using,including but not limited to, DEAE SEPHAROSE); chromatography on silica;high performance liquid chromatography (HPLC); reverse phase HPLC; gelfiltration (using, including but not limited to, SEPHADEX G-75);hydrophobic interaction chromatography; size-exclusion chromatography;metal-chelate chromatography; ultrafiltration/diafiltration; ethanolprecipitation; ammonium sulfate precipitation; chromatofocusing;displacement chromatography; electrophoretic procedures (including butnot limited to preparative isoelectric focusing), differentialsolubility (including but not limited to ammonium sulfateprecipitation), SDS-PAGE, or extraction.

Proteins of the present invention, including but not limited to,proteins comprising unnatural amino acids, antibodies to proteinscomprising unnatural amino acids, binding partners for proteinscomprising unnatural amino acids, etc., can be purified, eitherpartially or substantially to homogeneity, according to standardprocedures known to and used by those of skill in the art. Accordingly,polypeptides of the invention can be recovered and purified by any of anumber of methods known to those of ordinary skill in the art, includingbut not limited to, ammonium sulfate or ethanol precipitation, acid orbase extraction, column chromatography, affinity column chromatography,anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, hydroxylapatitechromatography, lectin chromatography, gel electrophoresis and the like.Protein refolding steps can be used, as desired, in making correctlyfolded mature proteins. High performance liquid chromatography (HPLC),affinity chromatography or other suitable methods can be employed infinal purification steps where high purity is desired. In oneembodiment, antibodies made against unnatural amino acids (or proteinscomprising unnatural amino acids) are used as purification reagents,including but not limited to, for affinity-based purification ofproteins comprising one or more unnatural amino acid(s). Once purified,partially or to homogeneity, as desired, the polypeptides are optionallyused for a wide variety of utilities, including but not limited to, asassay components, therapeutics, prophylaxis, diagnostics, researchreagents, and/or as immunogens for antibody production.

In addition to other references noted herein, a variety ofpurification/protein folding methods are known to those of ordinaryskill in the art, including, but not limited to, those set forth in R.Scopes, Protein Purification, Springer-Verlag, N.Y. (1982); Deutscher,Methods in Enzymology Vol. 182: Guide to Protein Purification, AcademicPress, Inc. N.Y. (1990); Sandana, (1997) Bioseparation of Proteins,Academic Press, Inc.; Bollag et al. (1996) Protein Methods, 2nd EditionWiley-Liss, N Y; Walker, (1996) The Protein Protocols Handbook HumanaPress, NJ, Harris and Angal, (1990) Protein Purification Applications: APractical Approach IRL Press at Oxford, Oxford, England; Harris andAngal, Protein Purification Methods: A Practical Approach IRL Press atOxford, Oxford, England; Scopes, (1993) Protein Purification: Principlesand Practice 3rd Edition Springer Verlag, NY; Janson and Ryden, (1998)Protein Purification Principles, High Resolution Methods andApplications, Second Edition Wiley-VCH, NY; and Walker (1998), ProteinProtocols on CD-ROM Humana Press, NJ; and the references cited therein.

One advantage of producing a protein or polypeptide of interest with anunnatural amino acid in a eukaryotic host cell or non-eukaryotic hostcell is that typically the proteins or polypeptides will be folded intheir native conformations. However, in certain embodiments of theinvention, those of skill in the art will recognize that, aftersynthesis, expression and/or purification, proteins can possess aconformation different from the desired conformations of the relevantpolypeptides. In one aspect of the invention, the expressed protein isoptionally denatured and then renatured. This is accomplished utilizingmethods known in the art, including but not limited to, by adding achaperonin to the protein or polypeptide of interest, by solubilizingthe proteins in a chaotropic agent such as guanidine HCl, utilizingprotein disulfide isomerase, etc.

In general, it is occasionally desirable to denature and reduceexpressed polypeptides and then to cause the polypeptides to re-foldinto the preferred conformation. For example, guanidine, urea, DTT, DTE,and/or a chaperonin can be added to a translation product of interest.Methods of reducing, denaturing and renaturing proteins are known tothose of ordinary skill in the art (see, the references above, andDebinski, et al. (1993) J. Biol. Chem., 268: 14065-14070; Kreitman andPastan (1993) Bioconjug. Chem., 4: 581-585; and Buchner, et al., (1992)Anal. Biochem., 205: 263-270). Debinski, et al., for example, describethe denaturation and reduction of inclusion body proteins inguanidine-DTE. The proteins can be refolded in a redox buffercontaining, including but not limited to, oxidized glutathione andL-arginine. Refolding reagents can be flowed or otherwise moved intocontact with the one or more polypeptide or other expression product, orvice-versa.

In the case of prokaryotic production of hGH, the hGH thus produced maybe misfolded and thus lack or have reduced biological activity. Thebioactivity of the protein may be restored by “refolding”. In general,misfolded hGH is refolded by solubilizing (where the hGH is alsoinsoluble), unfolding and reducing the polypeptide chain using, forexample, one or more chaotropic agents (e.g. urea and/or guanidine) anda reducing agent capable of reducing disulfide bonds (e.g.dithiothreitol, DTT or 2-mercaptoethanol, 2-ME). At a moderateconcentration of chaotrope, an oxidizing agent is then added (e.g.,oxygen, cystine or cystamine), which allows the reformation of disulfidebonds. hGH may be refolded using standard methods known in the art, suchas those described in U.S. Pat. Nos. 4,511,502, 4,511,503, and4,512,922.

After refolding, the hGH may be further purified. Purification of hGHmay be accomplished using a variety of techniques known to those ofordinary skill in the art, including hydrophobic interactionchromatography, size exclusion chromatography, ion exchangechromatography, reverse-phase high performance liquid chromatography,affinity chromatography, and the like or any combination thereof.Additional purification may also include a step of drying orprecipitation of the purified protein.

After purification, hGH may be exchanged into different buffers and/orconcentrated by any of a variety of methods known to the art, including,but not limited to, ultrafiltration, diafiltration and dialysis. hGHthat is provided as a single purified protein may be subject toaggregation and precipitation. A wide variety of materials to bufferexchange or concentrate polypeptides are known to those of ordinaryskill in the art.

The purified hGH may be at least 90% pure (as measured by reverse phasehigh performance liquid chromatography, RP-HPLC, or sodium dodecylsulfate-polyacrylamide gel electrophoresis, SDS-PAGE) or at least 95%pure, or at least 98% pure, or at least 99% or greater pure. Regardlessof the exact numerical value of the purity of the hGH, the hGH issufficiently pure for use as a pharmaceutical product or for furtherprocessing, such as conjugation with a water soluble polymer such asPEG.

Certain hGH molecules may be used as therapeutic agents in the absenceof other active ingredients or proteins (other than excipients,carriers, and stabilizers, serum albumin and the like), or they may becomplexed with another protein or a polymer.

XIII. Purification Methods

Any one of a variety of isolation steps may be performed on the celllysate, extract, culture medium, inclusion bodies, periplasmic space ofthe host cells, cytoplasm of the host cells, or other material,comprising hGH or on any hGH mixtures resulting from any isolation stepsincluding, but not limited to, affinity chromatography, ion exchangechromatography, hydrophobic interaction chromatography, gel filtrationchromatography, high performance liquid chromatography (“HPLC”),reversed phase-HPLC (“RP-HPLC”), expanded bed adsorption, or anycombination and/or repetition thereof and in any appropriate order.

Equipment and other necessary materials used in performing thetechniques described herein are commercially available. Pumps, fractioncollectors, monitors, recorders, and entire systems are available from,for example, Applied Biosystems (Foster City, Calif.), Bio-RadLaboratories, Inc. (Hercules, Calif.), and GE Healthcare, Inc.(Piscataway, N.J.). Chromatographic materials including, but not limitedto, exchange matrix materials, media, and buffers are also availablefrom such companies.

Equilibration, and other steps in the column chromatography processesdescribed herein such as washing and elution, may be more rapidlyaccomplished using specialized equipment such as a pump. Commerciallyavailable pumps include, but are not limited to, HILOAD® Pump P-50,Peristaltic Pump P-1, Pump P-901, and Pump P-903 (GE Healthcare,Piscataway, N.J.).

Examples of fraction collectors include RediFrac Fraction Collector,FRAC-100 and FRAC-200 Fraction Collectors, and SUPERFRAC® FractionCollector (GE Healthcare, Piscataway, N.J.). Mixers are also availableto form pH and linear concentration gradients. Commercially availablemixers include Gradient Mixer GM-1 and In-Line Mixers (GE Healthcare,Piscataway, N.J.).

The chromatographic process may be monitored using any commerciallyavailable monitor. Such monitors may be used to gather information likeUV, pH, and conductivity. Examples of detectors include Monitor UV-1,UVICORD® S II, Monitor UV-M II, Monitor UV-900, Monitor UPC-900, MonitorpH/C-900, and Conductivity Monitor (GE Healthcare, Piscataway, N.J.).Indeed, entire systems are commercially available including the variousAKTA® systems from GE Healthcare (Piscataway, N.J.).

As stated herein, the pH of the first hGH mixture may be adjusted priorto performing any subsequent isolation steps. In addition, the first hGHmixture or any subsequent mixture thereof may be concentrated usingtechniques known in the art. Moreover, the elution buffer comprising thefirst hGH mixture or any subsequent mixture thereof may be exchanged fora buffer suitable for the next isolation step using techniques known tothose of ordinary skill in the art.

Ion Exchange Chromatography

In one embodiment, and as an optional, additional step, ion exchangechromatography may be performed on the first hGH mixture. See generallyION EXCHANGE CHROMATOGRAPHY: PRINCIPLES AND METHODS (Cat. No.18-1114-21, GE Healthcare (Piscataway, N.J.)). Commercially availableion exchange columns include HITRAP®, HIPREP®, and HILOAD® Columns (GEHealthcare, Piscataway, N.J.). Such columns utilize strong anionexchangers such as Q SEPHAROSE® Fast Flow, Q SEPHAROSE® HighPerformance, and Q SEPHAROSE® XL; strong cation exchangers such as SPSEPHAROSE® High Performance, SP SEPHAROSE® Fast Flow, and SP SEPHAROSE®XL; weak anion exchangers such as DEAE SEPHAROSE® Fast Flow; and weakcation exchangers such as CM SEPHAROSE® Fast Flow (GE Healthcare,Piscataway, N.J.). Anion or cation exchange column chromatography may beperformed on the hGH at any stage of the purification process to isolatesubstantially purified hGH. Source 30Q and Source 30S are ion exchangemedia (GE Healthcare).

The cation exchange chromatography step may be performed using anysuitable cation exchange matrix. Useful cation exchange matricesinclude, but are not limited to, fibrous, porous, non-porous,microgranular, beaded, or cross-linked cation exchange matrix materials.Such cation exchange matrix materials include, but are not limited to,cellulose, agarose, dextran, polyacrylate, polyvinyl, polystyrene,silica, polyether, or composites of any of the foregoing.

The cation exchange matrix may be any suitable cation exchangerincluding strong and weak cation exchangers. Strong cation exchangersmay remain ionized over a wide pH range and thus, may be capable ofbinding hGH over a wide pH range. Weak cation exchangers, however, maylose ionization as a function of pH. For example, a weak cationexchanger may lose charge when the pH drops below about pH 4 or pH 5.Suitable strong cation exchangers include, but are not limited to,charged functional groups such as sulfopropyl (SP), methyl sulfonate(S), or sulfoethyl (SE). The cation exchange matrix may be a strongcation exchanger, having an hGH binding pH range of about 2.5 to about6.0. Alternatively, the strong cation exchanger may have an hGH bindingpH range of about pH 2.5 to about pH 5.5. The cation exchange matrix maybe a strong cation exchanger having an hGH binding pH of about 3.0.Alternatively, the cation exchange matrix may be a strong cationexchanger, having an hGH binding pH range of about 6.0 to about 8.0. Thecation exchange matrix may be a strong cation exchanger having an hGHbinding pH range of about 8.0 to about 12.5. Alternatively, the strongcation exchanger may have an hGH binding pH range of about pH 8.0 toabout pH 12.0.

Prior to loading the hGH, the cation exchange matrix may beequilibrated, for example, using several column volumes of a dilute,weak acid, e.g., four column volumes of 20 mM acetic acid, pH 3.Following equilibration, the hGH may be added and the column may bewashed one to several times, prior to elution of substantially purifiedhGH, also using a weak acid solution such as a weak acetic acid orphosphoric acid solution. For example, approximately 2-4 column volumesof 20 mM acetic acid, pH 3, may be used to wash the column. Additionalwashes using, e.g., 2-4 column volumes of 0.05 M sodium acetate, pH 5.5,or 0.05 M sodium acetate mixed with 0.1 M sodium chloride, pH 5.5, mayalso be used. Alternatively, using methods known in the art, the cationexchange matrix may be equilibrated using several column volumes of adilute, weak base.

Alternatively, substantially purified hGH may be eluted by contactingthe cation exchanger matrix with a buffer having a sufficiently low pHor ionic strength to displace the hGH from the matrix. The pH of theelution buffer may range from about pH 2.5 to about pH 6.0. Morespecifically, the pH of the elution buffer may range from about pH 2.5to about pH 5.5, about pH 2.5 to about pH 5.0. The elution buffer mayhave a pH of about 3.0. In addition, the quantity of elution buffer mayvary widely and will generally be in the range of about 2 to about 10column volumes. Moreover, suitable buffers known to those of skill inthe art may find use herein including, but not limited to, citrate,phosphate, formate, HEPES, and MES buffers ranging in concentration fromat least about 5 mM to at least about 100 mM.

Following adsorption of the hGH to the cation exchanger matrix,substantially purified hGH may be eluted by contacting the matrix with abuffer having a sufficiently high pH or ionic strength to displace thehGH from the matrix. The pH of the elution buffer may range from aboutpH 8.0 to about pH 12.5. More specifically, the elution buffer may rangefrom about pH 8.0 to about pH 12.0. Suitable buffers for use in high pHelution of substantially purified hGH include, but are not limited to,citrate, phosphate, formate, acetate, HEPES, and MES buffers ranging inconcentration from at least about 5 mM to at least about 100 mM. Inaddition, a buffer having 0.1 M potassium borate, 0.6 M potassiumchloride, 0.1 mM EDTA, pH 8.7 may be used. Substantially purified hGHmay also be eluted using standard buffers, such as a bicine buffer whichincludes about 50 to 100 mM bicine, about 75 mM bicine; 25 to about 100mM sodium chloride, specifically, about 50 mM sodium chloride, and about0.05 to about 0.5 EDTA, more specifically, about 0.1 mM EDTA, pH 7.5.

Reverse-Phase Chromatography

RP-HPLC may be performed to purify proteins following suitable protocolsthat are known to those of ordinary skill in the art. See, e.g., Pearsonet al., ANAL BIOCHEM. (1982) 124:217-230 (1982); Rivier et al., J.CHROM. (1983) 268:112-119; Kunitani et al., J. CHROM. (1986)359:391-402. RP-HPLC may be performed on the hGH to isolatesubstantially purified hGH. In this regard, silica derivatized resinswith alkyl functionalities with a wide variety of lengths, including,but not limited to, at least about C₃ to at least about C₃₀, at leastabout C₃ to at least about C₂₀, or at least about C₃ to at least aboutC₁₈, resins may be used. Alternatively, a polymeric resin may be used.For example, TosoHaas Amberchrome CG1000sd resin may be used, which is astyrene polymer resin. Cyano or polymeric resins with a wide variety ofalkyl chain lengths may also be used. Furthermore, the RP-HPLC columnmay be washed with a solvent such as ethanol. The Source RP column isanother example of a RP-HPLC column.

A suitable elution buffer containing an ion pairing agent and an organicmodifier such as methanol, isopropanol, tetrahydrofuran, acetonitrile orethanol, may be used to elute the hGH from the RP-HPLC column. The mostcommonly used ion pairing agents include, but are not limited to, aceticacid, formic acid, perchloric acid, phosphoric acid, trifluoroaceticacid, heptafluorobutyric acid, triethylamine, tetramethylammonium,tetrabutylammonium, triethylammonium acetate. Elution may be performedusing one or more gradients or isocratic conditions, with gradientconditions preferred to reduce the separation time and to decrease peakwidth. Generally, the gradient may be from about 5% to about 80% (v/v),about 5% to about 75% (v/v), about 5% to about 70% (v/v), about 5% toabout 65% (v/v), about 5% to about 60% (v/v), about 5% to about 55%(v/v), or about 10% to about 50% (v/v) solvent in water. Another methodinvolves the use of two gradients with different solvent concentrationranges. Examples of suitable elution buffers for use herein may include,but are not limited to, ammonium acetate and acetonitrile solutions.

The hGH derived from a recombinant E. coli host may be further isolatedor purified by reverse-phase chromatography. The hGH may be isolated,for example, using a SOURCE RP column, with an acetonitrile gradientfrom about 10% to about 60% acetonitrile.

Hydrophobic Interaction Chromatography Purification Techniques

Hydrophobic interaction chromatography (HIC) may be performed on thehGH. See generally HYDROPHOBIC INTERACTION CHROMATOGRAPHY HANDBOOK:PRINCIPLES AND METHODS (Cat. No. 18-1020-90, GE Healthcare (Piscataway,N.J.) which is incorporated by reference herein. Suitable HIC matricesmay include, but are not limited to, alkyl- or aryl-substitutedmatrices, such as butyl-, hexyl-, octyl- or phenyl-substituted matricesincluding agarose, cross-linked agarose, sepharose, cellulose, silica,dextran, polystyrene, poly(methacrylate) matrices, and mixed moderesins, including but not limited to, a polyethyleneamine resin or abutyl- or phenyl-substituted poly(methacrylate) matrix. Commerciallyavailable sources for hydrophobic interaction column chromatographyinclude, but are not limited to, HITRAP®, HIPREP®, and HILOAD® columns(GE Healthcare, Piscataway, N.J.), and TSKgel Phenyl-650S andPhenyl-5PW(30 um) resins (Tosoh Bioscience).

Briefly, prior to loading, the HIC column may be equilibrated usingstandard buffers known to those of ordinary skill in the art, such as anacetic acid/sodium chloride solution or HEPES containing ammoniumsulfate, or ammonium sulfate in a pH 6.5 sodium phosphate solution, orsodium sulfate in a pH 7-8 TRIS solution. Ammonium sulfate may be usedas the buffer for loading the HIC column. After loading the hGH, thecolumn may then washed using standard buffers and under conditions suchas those described herein to remove unwanted materials but retaining thehGH on the HIC column. hGH may be eluted with about 3 to about 10 columnvolumes of a standard buffer, such as a HEPES buffer containing EDTA andlower ammonium sulfate concentration than the equilibrating buffer, oran acetic acid/sodium chloride buffer, among others. A decreasing linearsalt gradient using, for example, a gradient of potassium phosphate, mayalso be used to elute the hGH molecules. Elution enhancers may also beadded to the elution buffer, including but not limited to, ethyleneglycol, glycerol, or urea (0.5-1.5M). The eluant may then beconcentrated, for example, by filtration such as diafiltration orultrafiltration. Diafiltration may be utilized to remove the salt usedto elute hGH.

Other Purification Techniques

Yet another isolation step using, for example, gel filtration (GELFILTRATION: PRINCIPLES AND METHODS (Cat. No. 18-1022-18, GE Healthcare,Piscataway, N.J.) which is incorporated by reference herein,hydroxyapatite chromatography (suitable matrices include, but are notlimited to, HA-Ultrogel, High Resolution (Calbiochem), CHT CeramicHydroxyapatite (BioRad), Bio—Gel HTP Hydroxyapatite (BioRad)), HPLC,expanded bed adsorption, ultrafiltration, diafiltration, lyophilization,and the like, may be performed on the first hGH mixture or anysubsequent mixture thereof, to remove any excess salts and to replacethe buffer with a suitable buffer for the next isolation step or evenformulation of the final drug product.

The non-naturally encoded amino acid present in the hGH molecule mayalso be utilized to provide separation from other cellular proteins thatdo not contain the non-naturally encoded amino acid. Since thenon-naturally encoded amino acid may comprise unique chemical functionalgroups, the coupling of the unique functional group to another moleculemay provide a substantial purification step. For example, thenon-naturally encoded amino acid may be coupled to another molecule thatfacilitates separation from other proteins. Such molecules for couplingto the non-natural amino acid include, but are not limited to, PEG andother polymers.

The yield of hGH, including substantially purified hGH, may be monitoredat each step described herein using techniques known to those ofordinary skill in the art. Such techniques may also be used to assessthe yield of substantially purified hGH following the last isolationstep. For example, the yield of hGH may be monitored using any ofseveral reverse phase high pressure liquid chromatography columns,having a variety of alkyl chain lengths such as cyano RP-HPLC,C₁₈RP-HPLC; as well as cation exchange HPLC and gel filtration HPLC.

In specific embodiments of the present invention, the yield of hGH aftereach purification step may be at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about55%, at least about 60%, at least about 65%, at least about 70%, atleast about 75%, at least about 80%, at least about 856%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, at least about 99%, at least about 99.9%, or atleast about 99.99%, of the hGH in the starting material for eachpurification step.

Purity may be determined using standard techniques, such as SDS-PAGE, orby measuring hGH using Western blot and ELISA assays. For example,polyclonal antibodies may be generated against proteins isolated from anegative control yeast fermentation and the cation exchange recovery.The antibodies may also be used to probe for the presence ofcontaminating host cell proteins.

RP-HPLC material Vydac C4 (Vydac) consists of silica gel particles, thesurfaces of which carry C4-alkyl chains. The separation of polypeptidesfrom the proteinaceous impurities is based on differences in thestrength of hydrophobic interactions. Elution is performed with anacetonitrile gradient in diluted trifluoroacetic acid. Preparative HPLCis performed using a stainless steel column (filled with 2.8 to 3.2liter of Vydac C4 silicagel). The Hydroxyapatite Ultrogel eluate isacidified by adding trifluoroacetic acid and loaded onto the Vydac C4column. For washing and elution an acetonitrile gradient in dilutedtrifluoroacetic acid is used. Fractions are collected and immediatelyneutralized with phosphate buffer. The polypeptide fractions which arewithin the IPC limits are pooled.

DEAE Sepharose (GE Healthcare) material consists of diethylaminoethyl(DEAE)-groups which are covalently bound to the surface of Sepharosebeads. The binding of a polypeptide of choice to the DEAE groups ismediated by ionic interactions. Acetonitrile and trifluoroacetic acidpass through the column without being retained. After these substanceshave been washed off, trace impurities are removed by washing the columnwith acetate buffer at a low pH. Then the column is washed with neutralphosphate buffer and polypeptide is eluted with a buffer with increasedionic strength. The column is packed with DEAE Sepharose fast flow. Thecolumn volume is adjusted to assure a polypeptide load in the range of3-10 mg polypeptide/ml gel. The column is washed with water andequilibration buffer (sodium/potassium phosphate). The pooled fractionsof the HPLC eluate are loaded and the column is washed withequilibration buffer. Then the column is washed with washing buffer(sodium acetate buffer) followed by washing with equilibration buffer.Subsequently, polypeptide is eluted from the column with elution buffer(sodium chloride, sodium/potassium phosphate) and collected in a singlefraction in accordance with the master elution profile. The eluate ofthe DEAE Sepharose column is adjusted to the specified conductivity. Theresulting drug substance is sterile filtered into Teflon bottles andstored at −70° C.

Methods and procedures that can be used to assess the yield and purityof hGH include, but are not limited to, the Bradford assay, SDS-PAGE,silver stained SDS-PAGE, coomassie stained SDS-PAGE, mass spectrometry(including but not limited to, MALDI-TOF) and other methods forcharacterizing proteins known to one of ordinary skill in the art.Additional methods include, but are not limited to: SDS-PAGE coupledwith protein staining methods, immunoblotting, matrix assisted laserdesorption/ionization-mass spectrometry (MALDI-MS), liquidchromatography/mass spectrometry, isoelectric focusing, analytical anionexchange, chromatofocusing, and circular dichroism.

Additional methods that may be employed include the steps to remove ofendotoxins. Endotoxins are lipopoly-saccharides (LPSs) which are locatedon the outer membrane of Gram-negative host cells, such as, for example,Escherichia coli. Methods for reducing endotoxin levels are known to oneof ordinary skill in the art and include, but are not limited to,purification techniques using silica supports, glass powder orhydroxyapatite, reverse-phase, affinity, size-exclusion, anion-exchangechromatography, hydrophobic interaction chromatography, filtration, acombination of these methods, and the like. Modifications or additionalmethods may be required to remove contaminants such as co-migratingproteins from the polypeptide of interest. Methods for measuringendotoxin levels are known to one of ordinary skill in the art andinclude, but are not limited to, Limulus Amebocyte Lysate (LAL) assays.

Although the invention has been described with reference to particularembodiments, methods, construction, and use, it will be apparent tothose of ordinary skill in the art that various changes andmodifications can be made without departing from the invention.Alterations to the reagents, materials, and purification conditionsindicated are apparent to those of ordinary skill in the art. Forexample, a higher capacity resin may be substituted in a chromatographystep if such capacity is desired.

XIV. Expression in Alternate Systems

Several strategies have been employed to introduce unnatural amino acidsinto proteins in non-recombinant host cells, mutagenized host cells, orin cell-free systems. These systems are also suitable for use in makingthe hGH polypeptides of the present invention. Derivatization of aminoacids with reactive side-chains such as Lys, Cys and Tyr resulted in theconversion of lysine to N²-acetyl-lysine. Chemical synthesis alsoprovides a straightforward method to incorporate unnatural amino acids.With the recent development of enzymatic ligation and native chemicalligation of peptide fragments, it is possible to make larger proteins.See, e.g., P. E. Dawson and S. B. H. Kent, Annu. Rev. Biochem, 69:923(2000). Chemical peptide ligation and native chemical ligation aredescribed in U.S. Pat. No. 6,184,344, U.S. Patent Publication No.2004/0138412, U.S. Patent Publication No. 2003/0208046, WO 02/098902,and WO 03/042235, which are incorporated by reference herein. A generalin vitro biosynthetic method in which a suppressor tRNA chemicallyacylated with the desired unnatural amino acid is added to an in vitroextract capable of supporting protein biosynthesis, has been used tosite-specifically incorporate over 100 unnatural amino acids into avariety of proteins of virtually any size. See, e.g., V. W. Cornish, D.Mendel and P. G. Schultz, Angew. Chem. Int. Ed. Engl., 1995, 34:621(1995); C. J. Noren, S. J. Anthony-Cahill, M. C. Griffith, P. G.Schultz, A general method for site-specific incorporation of unnaturalamino acids into proteins, Science 244:182-188 (1989); and, J. D. Bain,C. G. Glabe, T. A. Dix, A. R. Chamberlin, E. S. Diala, Biosyntheticsite-specific incorporation of a non-natural amino acid into apolypeptide, J. Am. Chem. Soc. 111:8013-8014 (1989). A broad range offunctional groups has been introduced into proteins for studies ofprotein stability, protein folding, enzyme mechanism, and signaltransduction.

An in vivo method, termed selective pressure incorporation, wasdeveloped to exploit the promiscuity of wild-type synthetases. See,e.g., N. Budisa, C. Minks, S. Alefelder, W. Wenger, F. M. Dong, L.Moroder and R. Huber, FASEB J., 13:41 (1999). An auxotrophic strain, inwhich the relevant metabolic pathway supplying the cell with aparticular natural amino acid is switched off, is grown in minimal mediacontaining limited concentrations of the natural amino acid, whiletranscription of the target gene is repressed. At the onset of astationary growth phase, the natural amino acid is depleted and replacedwith the unnatural amino acid analog. Induction of expression of therecombinant protein results in the accumulation of a protein containingthe unnatural analog. For example, using this strategy, o, m andp-fluorophenylalanines have been incorporated into proteins, and exhibittwo characteristic shoulders in the UV spectrum which can be easilyidentified, see, e.g., C. Minks, R. Huber, L. Moroder and N. Budisa,Anal. Biochem., 284:29 (2000); trifluoromethionine has been used toreplace methionine in bacteriophage T4 lysozyme to study its interactionwith chitooligosaccharide ligands by ¹⁹F NMR, see, e.g., H. Duewel, E.Daub, V. Robinson and J. F. Honek, Biochemistry, 36:3404 (1997); andtrifluoroleucine has been incorporated in place of leucine, resulting inincreased thermal and chemical stability of a leucine-zipper protein.See, e.g., Y. Tang, G. Ghirlanda, W. A. Petka, T. Nakajima, W. F.DeGrado and D. A. Tirrell, Angew. Chem. Int. Ed. Engl., 40:1494 (2001).Moreover, selenomethionine and telluromethionine are incorporated intovarious recombinant proteins to facilitate the solution of phases inX-ray crystallography. See, e.g., W. A. Hendrickson, J. R. Horton and D.M. Lemaster, EMBO J., 9:1665 (1990); J. O. Boles, K. Lewinski, M.Kunkle, J. D. Odom, B. Dunlap, L. Lebioda and M. Hatada, Nat. Struct.Biol., 1:283 (1994); N. Budisa, B. Steipe, P. Demange, C. Eckerskorn, J.Kellermann and R. Huber, Eur. J. Biochem., 230:788 (1995); and, N.Budisa, W. Kambrock, S. Steinbacher, A. Humm, L. Prade, T. Neuefeind, L.Moroder and R. Huber, J. Mol. Biol., 270:616 (1997). Methionine analogswith alkene or alkyne functionalities have also been incorporatedefficiently, allowing for additional modification of proteins bychemical means. See, e.g., J. C. van Hest and D. A. Tirrell, FEBS Lett.,428:68 (1998); J. C. van Hest, K. L. Kiick and D. A. Tirrell, J. Am.Chem. Soc., 122:1282 (2000); and, K. L. Kiick and D. A. Tirrell,Tetrahedron, 56:9487 (2000); U.S. Pat. No. 6,586,207; U.S. PatentPublication 2002/0042097, which are incorporated by reference herein.

The success of this method depends on the recognition of the unnaturalamino acid analogs by aminoacyl-tRNA synthetases, which, in general,require high selectivity to insure the fidelity of protein translation.One way to expand the scope of this method is to relax the substratespecificity of aminoacyl-tRNA synthetases, which has been achieved in alimited number of cases. For example, replacement of Ala²⁹⁴ by Gly inEscherichia coli phenylalanyl-tRNA synthetase (PheRS) increases the sizeof substrate binding pocket, and results in the acylation of tRNAPhe byp-Cl-phenylalanine (p-Cl-Phe). See, M. Ibba, P. Kast and H. Hennecke,Biochemistry, 33:7107 (1994). An Escherichia coli strain harboring thismutant PheRS allows the incorporation of p-Cl-phenylalanine orp-Br-phenylalanine in place of phenylalanine. See, e.g., M. Ibba and H.Hennecke, FEBS Lett., 364:272 (1995); and, N. Sharma, R. Furter, P. Kastand D. A. Tirrell, FEBS Lett., 467:37 (2000). Similarly, a pointmutation Phe130Ser near the amino acid binding site of Escherichia colityrosyl-tRNA synthetase was shown to allow azatyrosine to beincorporated more efficiently than tyrosine. See, F. Hamano-Takaku, T.Iwama, S. Saito-Yano, K. Takaku, Y. Monden, M. Kitabatake, D. Soll andS, Nishimura, J. Biol. Chem., 275:40324 (2000).

Another strategy to incorporate unnatural amino acids into proteins invivo is to modify synthetases that have proofreading mechanisms. Thesesynthetases cannot discriminate and therefore activate amino acids thatare structurally similar to the cognate natural amino acids. This erroris corrected at a separate site, which deacylates the mischarged aminoacid from the tRNA to maintain the fidelity of protein translation. Ifthe proofreading activity of the synthetase is disabled, structuralanalogs that are misactivated may escape the editing function and beincorporated. This approach has been demonstrated recently with thevalyl-tRNA synthetase (ValRS). See, V. Doring, H. D. Mootz, L. A.Nangle, T. L. Hendrickson, V. de Crecy-Lagard, P. Schimmel and P.Marliere, Science, 292:501 (2001). ValRS can misaminoacylate tRNAValwith Cys; Thr, or aminobutyrate (Abu); these noncognate amino acids aresubsequently hydrolyzed by the editing domain. After random mutagenesisof the Escherichia coli chromosome, a mutant Escherichia coli strain wasselected that has a mutation in the editing site of ValRS. Thisedit-defective ValRS incorrectly charges tRNAVal with Cys. Because Abusterically resembles Cys (—SH group of Cys is replaced with —CH3 inAbu), the mutant ValRS also incorporates Abu into proteins when thismutant Escherichia coli strain is grown in the presence of Abu. Massspectrometric analysis shows that about 24% of valines are replaced byAbu at each valine position in the native protein.

Solid-phase synthesis and semisynthetic methods have also allowed forthe synthesis of a number of proteins containing novel amino acids. Forexample, see the following publications and references cited within,which are as follows: Crick, F. H. C., Barrett, L. Brenner, S.Watts-Tobin, R. General nature of the genetic code for proteins. Nature,192:1227-1232 (1961); Hofmann, K., Bohn, H. Studies on polypeptides.XXXVI. The effect of pyrazole-imidazole replacements on the S-proteinactivating potency of an S-peptide fragment, J. Am. Chem,88(24):5914-5919 (1966); Kaiser, E. T. Synthetic approaches tobiologically active peptides and proteins including enyzmes, Acc ChemRes, 22:47-54 (1989); Nakatsuka, T., Sasaki, T., Kaiser, E. T. Peptidesegment coupling catalyzed by the semisynthetic enzyme thiosubtilisin, JAm Chem Soc, 109:3808-3810 (1987); Schnolzer, M., Kent, S B H.Constructing proteins by dovetailing unprotected synthetic peptides:backbone-engineered HIV protease, Science, 256(5054):221-225 (1992);Chaiken, I. M. Semisynthetic peptides and proteins, CRC Crit RevBiochem, 11(3):255-301 (1981); Offord, R. E. Protein engineering bychemical means? Protein Eng., 1(3):151-157 (1987); and, Jackson, D. Y.,Burnier, J., Quan, C., Stanley, M., Tom, J., Wells, J. A. A DesignedPeptide Ligase for Total Synthesis of Ribonuclease A with UnnaturalCatalytic Residues, Science, 266(5183):243 (1994).

Chemical modification has been used to introduce a variety of unnaturalside chains, including cofactors, spin labels and oligonucleotides intoproteins in vitro. See, e.g., Corey, D. R., Schultz, P. G. Generation ofa hybrid sequence-specific single-stranded deoxyribonuclease, Science,238(4832):1401-1403 (1987); Kaiser, E. T., Lawrence D. S., Rokita, S. E.The chemical modification of enzymatic specificity, Annu Rev Biochem,54:565-595 (1985); Kaiser, E. T., Lawrence, D. S. Chemical mutation ofenyzme active sites, Science, 226(4674):505-511 (1984); Neet, K. E.,Nanci A, Koshland, D. E. Properties of thiol-subtilisin, J. Biol. Chem.,243(24):6392-6401 (1968); Polgar, L. et M. L. Bender. A new enzymecontaining a synthetically formed active site. Thiol-subtilisin. J. Am.Chem Soc, 88:3153-3154 (1966); and, Pollack, S. J., Nakayama, G.Schultz, P. G. Introduction of nucleophiles and spectroscopic probesinto antibody combining sites, Science, 242(4881):1038-1040 (1988).

Alternatively, biosynthetic methods that employ chemically modifiedaminoacyl-tRNAs have been used to incorporate several biophysical probesinto proteins synthesized in vitro. See the following publications andreferences cited within: Brunner, J. New Photolabeling and crosslinkingmethods, Annu. Rev Biochem, 62:483-514 (1993); and, Krieg, U. C.,Walter, P., Hohnson, A. E. Photocrosslinking of the signal sequence ofnascent preprolactin of the 54-kilodalton polypeptide of the signalrecognition particle, Proc. Natl. Acad. Sci, 83(22):8604-8608 (1986).

Previously, it has been shown that unnatural amino acids can besite-specifically incorporated into proteins in vitro by the addition ofchemically aminoacylated suppressor tRNAs to protein synthesis reactionsprogrammed with a gene containing a desired amber nonsense mutation.Using these approaches, one can substitute a number of the common twentyamino acids with close structural homologues, e.g., fluorophenylalaninefor phenylalanine, using strains auxotropic for a particular amino acid.See, e.g., Noren, C. J., Anthony-Cahill, Griffith, M. C., Schultz, P. G.A general method for site-specific incorporation of unnatural aminoacids into proteins, Science, 244: 182-188 (1989); M. W. Nowak, et al.,Science 268:439-42 (1995); Bain, J. D., Glabe, C. G., Dix, T. A.,Chamberlin, A. R., Diala, E. S. Biosynthetic site-specific Incorporationof a non-natural amino acid into a polypeptide, J. Am. Chem Soc,111:8013-8014 (1989); N. Budisa et al., FASEB J. 13:41-51 (1999);Ellman, J. A., Mendel, D., Anthony-Cahill, S., Noren, C. J., Schultz, P.G. Biosynthetic method for introducing unnatural amino acidssite-specifically into proteins, Methods in Enz., vol. 202, 301-336(1992); and, Mendel, D., Cornish, V. W. & Schultz, P. G. Site-DirectedMutagenesis with an Expanded Genetic Code, Annu Rev Biophys. BiomolStruct. 24, 435-62 (1995).

For example, a suppressor tRNA was prepared that recognized the stopcodon UAG and was chemically aminoacylated with an unnatural amino acid.Conventional site-directed mutagenesis was used to introduce the stopcodon TAG, at the site of interest in the protein gene. See, e.g.,Sayers, J. R., Schmidt, W. Eckstein, F. 5′-3′ Exonucleases inphosphorothioate-based olignoucleotide-directed mutagensis, NucleicAcids Res, 16(3):791-802 (1988). When the acylated suppressor tRNA andthe mutant gene were combined in an in vitro transcription/translationsystem, the unnatural amino acid was incorporated in response to the UAGcodon which gave a protein containing that amino acid at the specifiedposition. Experiments using [³H]-Phe and experiments with α-hydroxyacids demonstrated that only the desired amino acid is incorporated atthe position specified by the UAG codon and that this amino acid is notincorporated at any other site in the protein. See, e.g., Noren, et al,supra; Kobayashi et al., (2003) Nature Structural Biology 10(6):425-432;and, Ellman, J. A., Mendel, D., Schultz, P. G. Site-specificincorporation of novel backbone structures into proteins, Science,255(5041):197-200 (1992).

A tRNA may be aminoacylated with a desired amino acid by any method ortechnique, including but not limited to, chemical or enzymaticaminoacylation.

Aminoacylation may be accomplished by aminoacyl tRNA synthetases or byother enzymatic molecules, including but not limited to, ribozymes. Theterm “ribozyme” is interchangeable with “catalytic RNA.” Cech andcoworkers (Cech, 1987, Science, 236:15327 1539; McCorkle et al., 1987,Concepts Biochem. 64:221-226) demonstrated the presence of naturallyoccurring RNAs that can act as catalysts (ribozymes). However, althoughthese natural RNA catalysts have only been shown to act on ribonucleicacid substrates for cleavage and splicing, the recent development ofartificial evolution of ribozymes has expanded the repertoire ofcatalysis to various chemical reactions. Studies have identified RNAmolecules that can catalyze aminoacyl-RNA bonds on their own(2′)₃′-termini (Illangakekare et al., 1995 Science 267:643-647), and anRNA molecule which can transfer an amino acid from one RNA molecule toanother (Lohse et al., 1996, Nature 381:442-444).

U.S. Patent Application Publication 2003/0228593, which is incorporatedby reference herein, describes methods to construct ribozymes and theiruse in aminoacylation of tRNAs with naturally encoded and non-naturallyencoded amino acids. Substrate-immobilized forms of enzymatic moleculesthat can aminoacylate tRNAs, including but not limited to, ribozymes,may enable efficient affinity purification of the aminoacylatedproducts. Examples of suitable substrates include agarose, sepharose,and magnetic beads. The production and use of a substrate-immobilizedform of ribozyme for aminoacylation is described in Chemistry andBiology 2003, 10:1077-1084 and U.S. Patent Application Publication2003/0228593, which are incorporated by reference herein.

Chemical aminoacylation methods include, but are not limited to, thoseintroduced by Hecht and coworkers (Hecht, S. M. Acc. Chem. Res. 1992,25, 545; Heckler, T. G.; Roesser, J. R.; Xu, C.; Chang, P.; Hecht, S. M.Biochemistry 1988, 27, 7254; Hecht, S. M.; Alford, B. L.; Kuroda, Y.;Kitano, S. J. Biol. Chem. 1978, 253, 4517) and by Schultz, Chamberlin,Dougherty and others (Cornish, V. W.; Mendel, D.; Schultz, P. G. Angew.Chem. Int. Ed. Engl. 1995, 34, 621; Robertson, S. A.; Ellman, J. A.;Schultz, P. G. J. Am. Chem. Soc. 1991, 113, 2722; Noren, C. J.;Anthony-Cahill, S. J.; Griffith, M. C.; Schultz, P. G. Science 1989,244, 182; Bain, J. D.; Glabe, C. G.; Dix, T. A.; Chamberlin, A. R. J.Am. Chem. Soc. 1989, 111, 8013; Bain, J. D. et al. Nature 1992, 356,537; Gallivan, J. P.; Lester, H. A.; Dougherty, D. A. Chem. Biol. 1997,4, 740; Turcatti, et al. J. Biol. Chem. 1996, 271, 19991; Nowak, M. W.et al. Science, 1995, 268, 439; Saks, M. E. et al. J. Biol. Chem. 1996,271, 23169; Hohsaka, T. et al. J. Am. Chem. Soc. 1999, 121, 34), whichare incorporated by reference herein, to avoid the use of synthetases inaminoacylation. Such methods or other chemical aminoacylation methodsmay be used to aminoacylate tRNA molecules.

Methods for generating catalytic RNA may involve generating separatepools of randomized ribozyme sequences, performing directed evolution onthe pools, screening the pools for desirable aminoacylation activity,and selecting sequences of those ribozymes exhibiting desiredaminoacylation activity.

Ribozymes can comprise motifs and/or regions that facilitate acylationactivity, such as a GGU motif and a U-rich region. For example, it hasbeen reported that U-rich regions can facilitate recognition of an aminoacid substrate, and a GGU-motif can form base pairs with the 3′ terminiof a tRNA. In combination, the GGU and motif and U-rich regionfacilitate simultaneous recognition of both the amino acid and tRNAsimultaneously, and thereby facilitate aminoacylation of the 3′ terminusof the tRNA.

Ribozymes can be generated by in vitro selection using a partiallyrandomized r24 mini conjugated with tRNA^(ASn) _(CCCG), followed bysystematic engineering of a consensus sequence found in the activeclones. An exemplary ribozyme obtained by this method is termed “Fx3ribozyme” and is described in U.S. Pub. App. No. 2003/0228593, thecontents of which is incorporated by reference herein, acts as aversatile catalyst for the synthesis of various aminoacyl-tRNAs chargedwith cognate non-natural amino acids.

Immobilization on a substrate may be used to enable efficient affinitypurification of the aminoacylated tRNAs. Examples of suitable substratesinclude, but are not limited to, agarose, sepharose, and magnetic beads.Ribozymes can be immobilized on resins by taking advantage of thechemical structure of RNA, such as the 3′-cis-diol on the ribose of RNAcan be oxidized with periodate to yield the corresponding dialdehyde tofacilitate immobilization of the RNA on the resin. Various types ofresins can be used including inexpensive hydrazide resins whereinreductive amination makes the interaction between the resin and theribozyme an irreversible linkage. Synthesis of aminoacyl-tRNAs can besignificantly facilitated by this on-column aminoacylation technique.Kouroukli s et al. Methods 2005; 36:239-4 describe a column-basedaminoacylation system.

Isolation of the aminoacylated tRNAs can be accomplished in a variety ofways. One suitable method is to elute the aminoacylated tRNAs from acolumn with a buffer such as a sodium acetate solution with 10 mM EDTA,a buffer containing 50 mMN-(2-hydroxyethyl)piperazine-N′-(3-propanesulfonic acid), 12.5 mM KCl,pH 7.0, 10 mM EDTA, or simply an EDTA buffered water (pH 7.0).

The aminoacylated tRNAs can be added to translation reactions in orderto incorporate the amino acid with which the tRNA was aminoacylated in aposition of choice in a polypeptide made by the translation reaction.Examples of translation systems in which the aminoacylated tRNAs of thepresent invention may be used include, but are not limited to celllysates. Cell lysates provide reaction components necessary for in vitrotranslation of a polypeptide from an input mRNA. Examples of suchreaction components include but are not limited to ribosomal proteins,rRNA, amino acids, tRNAs, GTP, ATP, translation initiation andelongation factors and additional factors associated with translation.Additionally, translation systems may be batch translations orcompartmentalized translation. Batch translation systems combinereaction components in a single compartment while compartmentalizedtranslation systems separate the translation reaction components fromreaction products that can inhibit the translation efficiency. Suchtranslation systems are available commercially.

Further, a coupled transcription/translation system may be used. Coupledtranscription/translation systems allow for both transcription of aninput DNA into a corresponding mRNA, which is in turn translated by thereaction components. An example of a commercially available coupledtranscription/translation is the Rapid Translation System (RTS, RocheInc.). The system includes a mixture containing E. coli lysate forproviding translational components such as ribosomes and translationfactors. Additionally, an RNA polymerase is included for thetranscription of the input DNA into an mRNA template for use intranslation. RTS can use compartmentalization of the reaction componentsby way of a membrane interposed between reaction compartments, includinga supply/waste compartment and a transcription/translation compartment.

Aminoacylation of tRNA may be performed by other agents, including butnot limited to, transferases, polymerases, catalytic antibodies,multi-functional proteins, and the like.

Lu et al. in Mol. Cell. 2001 October; 8(4):759-69 describe a method inwhich a protein is chemically ligated to a synthetic peptide containingunnatural amino acids (expressed protein ligation).

Microinjection techniques have also been use incorporate unnatural aminoacids into proteins. See, e.g., M. W. Nowak, P. C. Kearney, J. R.Sampson, M. E. Saks, C. G. Labarca, S. K. Silverman, W. G. Zhong, J.Thorson, J. N. Abelson, N. Davidson, P. G. Schultz, D. A. Dougherty andH. A. Lester, Science, 268:439 (1995); and, D. A. Dougherty, Curr. Opin.Chem. Biol., 4:645 (2000). A Xenopus oocyte was coinjected with two RNAspecies made in vitro: an mRNA encoding the target protein with a UAGstop codon at the amino acid position of interest and an ambersuppressor tRNA aminoacylated with the desired unnatural amino acid. Thetranslational machinery of the oocyte then inserts the unnatural aminoacid at the position specified by UAG. This method has allowed in vivostructure-function studies of integral membrane proteins, which aregenerally not amenable to in vitro expression systems. Examples includethe incorporation of a fluorescent amino acid into tachykininneurokinin-2 receptor to measure distances by fluorescence resonanceenergy transfer, see, e.g., G. Turcatti, K. Nemeth, M. D. Edgerton, U.Meseth, F. Talabot, M. Peitsch, J. Knowles, H. Vogel and A. Chollet, J.Biol. Chem., 271:19991 (1996); the incorporation of biotinylated aminoacids to identify surface-exposed residues in ion channels, see, e.g.,J. P. Gallivan, H. A. Lester and D. A. Dougherty, Chem. Biol., 4:739(1997); the use of caged tyrosine analogs to monitor conformationalchanges in an ion channel in real time, see, e.g., J. C. Miller, S. K.Silverman, P. M. England, D. A. Dougherty and H. A. Lester, Neuron,20:619 (1998); and, the use of alpha hydroxy amino acids to change ionchannel backbones for probing their gating mechanisms. See, e.g., P. M.England, Y. Zhang, D. A. Dougherty and H. A. Lester, Cell, 96:89 (1999);and, T. Lu, A. Y. Ting, J. Mainland, L. Y. Jan, P. G. Schultz and J.Yang, Nat. Neurosci., 4:239 (2001).

The ability to incorporate unnatural amino acids directly into proteinsin vivo offers a wide variety of advantages of including but not limitedto, high yields of mutant proteins, technical ease, the potential tostudy the mutant proteins in cells or possibly in living organisms andthe use of these mutant proteins in therapeutic treatments anddiagnostic uses. The ability to include unnatural amino acids withvarious sizes, acidities, nucleophilicities, hydrophobicities, and otherproperties into proteins can greatly expand our ability to rationallyand systematically manipulate the structures of proteins, both to probeprotein function and create new proteins or organisms with novelproperties. However, the process is difficult, because the complexnature of tRNA-synthetase interactions that are required to achieve ahigh degree of fidelity in protein translation.

In one attempt to site-specifically incorporate para-F-Phe, a yeastamber suppressor tRNAPheCUA/phenylalanyl-tRNA synthetase pair was usedin a p-F-Phe resistant, Phe auxotrophic Escherichia coli strain. See,e.g., R. Furter, Protein Sci., 7:419 (1998).

It may also be possible to obtain expression of a hGH polynucleotide ofthe present invention using a cell-free (in-vitro) translational system.Translation systems may be cellular or cell-free, and may be prokaryoticor eukaryotic. Cellular translation systems include, but are not limitedto, whole cell preparations such as permeabilized cells or cell cultureswherein a desired nucleic acid sequence can be transcribed to mRNA andthe mRNA translated. Cell-free translation systems are commerciallyavailable and many different types and systems are well-known. Examplesof cell-free systems include, but are not limited to, prokaryoticlysates such as Escherichia coli lysates, and eukaryotic lysates such aswheat germ extracts, insect cell lysates, rabbit reticulocyte lysates,rabbit oocyte lysates and human cell lysates. Eukaryotic extracts orlysates may be preferred when the resulting protein is glycosylated,phosphorylated or otherwise modified because many such modifications areonly possible in eukaryotic systems. Some of these extracts and lysatesare available commercially (Promega; Madison, Wis.; Stratagene; LaJolla, Calif.; Amersham; Arlington Heights, Ill.; GIBCO/BRL; GrandIsland, N.Y.). Membranous extracts, such as the canine pancreaticextracts containing microsomal membranes, are also available which areuseful for translating secretory proteins. In these systems, which caninclude either mRNA as a template (in-vitro translation) or DNA as atemplate (combined in-vitro transcription and translation), the in vitrosynthesis is directed by the ribosomes. Considerable effort has beenapplied to the development of cell-free protein expression systems. See,e.g., Kim, D. M. and J. R. Swartz, Biotechnology and Bioengineering,74:309-316 (2001); Kim, D. M. and J. R. Swartz, Biotechnology Letters,22, 1537-1542, (2000); Kim, D. M., and J. R. Swartz, BiotechnologyProgress, 16, 385-390, (2000); Kim, D. M., and J. R. Swartz,Biotechnology and Bioengineering, 66, 180-188, (1999); and Patnaik, R.and J. R. Swartz, Biotechniques 24, 862-868, (1998); U.S. Pat. No.6,337,191; U.S. Patent Publication No. 2002/0081660; WO 00/55353; WO90/05785, which are incorporated by reference herein. Another approachthat may be applied to the expression of hGH polypeptides comprising anon-naturally encoded amino acid includes the mRNA-peptide fusiontechnique. See, e.g., R. Roberts and J. Szostak, Proc. Natl. Acad. Sci.(USA) 94:12297-12302 (1997); A. Frankel, et al., Chemistry & Biology10:1043-1050 (2003). In this approach, an mRNA template linked topuromycin is translated into peptide on the ribosome. If one or moretRNA molecules has been modified, non-natural amino acids can beincorporated into the peptide as well. After the last mRNA codon hasbeen read, puromycin captures the C-terminus of the peptide. If theresulting mRNA-peptide conjugate is found to have interesting propertiesin an in vitro assay, its identity can be easily revealed from the mRNAsequence. In this way, one may screen libraries of hGH polypeptidescomprising one or more non-naturally encoded amino acids to identifypolypeptides having desired properties. More recently, in vitro ribosometranslations with purified components have been reported that permit thesynthesis of peptides substituted with non-naturally encoded aminoacids. See, e.g., A. Forster et al., Proc. Natl. Acad. Sci. (USA)100:6353 (2003).

Reconstituted translation systems may also be used. Mixtures of purifiedtranslation factors have also been used successfully to translate mRNAinto protein as well as combinations of lysates or lysates supplementedwith purified translation factors such as initiation factor-1 (IF-1),IF-2, IF-3 (α or β), elongation factor T (EF-Tu), or terminationfactors. Cell-free systems may also be coupled transcription/translationsystems wherein DNA is introduced to the system, transcribed into mRNAand the mRNA translated as described in Current Protocols in MolecularBiology (F. M. Ausubel et al. editors, Wiley Interscience, 1993), whichis hereby specifically incorporated by reference. RNA transcribed ineukaryotic transcription system may be in the form of heteronuclear RNA(hnRNA) or 5′-end caps (7-methyl guanosine) and 3′-end poly A tailedmature mRNA, which can be an advantage in certain translation systems.For example, capped mRNAs are translated with high efficiency in thereticulocyte lysate system.

XV. Macromolecular Polymers Coupled to hGH Polypeptides

Various modifications to the non-natural amino acid polypeptidesdescribed herein can be effected using the compositions, methods,techniques and strategies described herein. These modifications includethe incorporation of further functionality onto the non-natural aminoacid component of the polypeptide, including but not limited to, alabel; a dye; a polymer; a water-soluble polymer; a derivative ofpolyethylene glycol; a photocrosslinker; a radionuclide; a cytotoxiccompound; a drug; an affinity label; a photoaffinity label; a reactivecompound; a resin; a second protein or polypeptide or polypeptideanalog; an antibody or antibody fragment; a metal chelator; a cofactor;a fatty acid; a carbohydrate; a polynucleotide; a DNA; a RNA; anantisense polynucleotide; a saccharide; a water-soluble dendrimer; acyclodextrin; an inhibitory ribonucleic acid; a biomaterial; ananoparticle; a spin label; a fluorophore, a metal-containing moiety; aradioactive moiety; a novel functional group; a group that covalently ornoncovalently interacts with other molecules; a photocaged moiety; anactinic radiation excitable moiety; a photoisomerizable moiety; biotin;a derivative of biotin; a biotin analogue; a moiety incorporating aheavy atom; a chemically cleavable group; a photocleavable group; anelongated side chain; a carbon-linked sugar; a redox-active agent; anamino thioacid; a toxic moiety; an isotopically labeled moiety; abiophysical probe; a phosphorescent group; a chemiluminescent group; anelectron dense group; a magnetic group; an intercalating group; achromophore; an energy transfer agent; a biologically active agent; adetectable label; a small molecule; a quantum dot; a nanotransmitter; aradionucleotide; a radiotransmitter; a neutron-capture agent; or anycombination of the above, or any other desirable compound or substance.As an illustrative, non-limiting example of the compositions, methods,techniques and strategies described herein, the following descriptionwill focus on adding macromolecular polymers to the non-natural aminoacid polypeptide with the understanding that the compositions, methods,techniques and strategies described thereto are also applicable (withappropriate modifications, if necessary and for which one of skill inthe art could make with the disclosures herein) to adding otherfunctionalities, including but not limited to those listed above.

A wide variety of macromolecular polymers and other molecules can belinked to hGH polypeptides of the present invention to modulatebiological properties of the hGH polypeptide, and/or provide newbiological properties to the hGH molecule. These macromolecular polymerscan be linked to the hGH polypeptide via a naturally encoded amino acid,via a non-naturally encoded amino acid, or any functional substituent ofa natural or non-natural amino acid, or any substituent or functionalgroup added to a natural or non-natural amino acid. The molecular weightof the polymer may be of a wide range, including but not limited to,between about 100 Da and about 100,000 Da or more. The molecular weightof the polymer may be between about 100 Da and about 100,000 Da,including but not limited to, 100,000 Da, 95,000 Da, 90,000 Da, 85,000Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da,50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000Da, 4,000 Da, 3,000 Da, 2,000 Da, 1,000 Da, 900 Da, 800 Da, 700 Da, 600Da, 500 Da, 400 Da, 300 Da, 200 Da, and 100 Da. In some embodiments, themolecular weight of the polymer is between about 100 Da and 50,000 Da.In some embodiments, the molecular weight of the polymer is betweenabout 100 Da and 40,000 Da. In some embodiments, the molecular weight ofthe polymer is between about 1,000 Da and 40,000 Da. In someembodiments, the molecular weight of the polymer is between about 5,000Da and 40,000 Da. In some embodiments, the molecular weight of thepolymer is between about 10,000 Da and 40,000 Da.

The present invention provides substantially homogenous preparations ofpolymer:protein conjugates. “Substantially homogenous” as used hereinmeans that polymer:protein conjugate molecules are observed to begreater than half of the total protein. The polymer:protein conjugatehas biological activity and the present “substantially homogenous”PEGylated hGH polypeptide preparations provided herein are those whichare homogenous enough to display the advantages of a homogenouspreparation, e.g., ease in clinical application in predictability of lotto lot pharmacokinetics.

One may also choose to prepare a mixture of polymer:protein conjugatemolecules, and the advantage provided herein is that one may select theproportion of mono-polymer:protein conjugate to include in the mixture.Thus, if desired, one may prepare a mixture of various proteins withvarious numbers of polymer moieties attached (i.e., di-, tri-, tetra-,etc.) and combine said conjugates with the mono-polymer:proteinconjugate prepared using the methods of the present invention, and havea mixture with a predetermined proportion of mono-polymer:proteinconjugates.

The polymer selected may be water soluble so that the protein to whichit is attached does not precipitate in an aqueous environment, such as aphysiological environment. The polymer may be branched or unbranched.For therapeutic use of the end-product preparation, the polymer will bepharmaceutically acceptable.

Examples of polymers include but are not limited to polyalkyl ethers andalkoxy-capped analogs thereof (e.g., polyoxyethylene glycol,polyoxyethylene/propylene glycol, and methoxy or ethoxy-capped analogsthereof, especially polyoxyethylene glycol, the latter is also known aspolyethyleneglycol or PEG); polyvinylpyrrolidones; polyvinylalkylethers; polyoxazolines, polyalkyl oxazolines and polyhydroxyalkyloxazolines; polyacrylamides, polyalkyl acrylamides, and polyhydroxyalkylacrylamides (e.g., polyhydroxypropylmethacrylamide and derivativesthereof); polyhydroxyalkyl acrylates; polysialic acids and analogsthereof; hydrophilic peptide sequences; polysaccharides and theirderivatives, including dextran and dextran derivatives, e.g.,carboxymethyldextran, dextran sulfates, aminodextran; cellulose and itsderivatives, e.g., carboxymethyl cellulose, hydroxyalkyl celluloses;chitin and its derivatives, e.g., chitosan, succinyl chitosan,carboxymethylchitin, carboxymethylchitosan; hyaluronic acid and itsderivatives; starches; alginates; chondroitin sulfate; albumin; pullulanand carboxymethyl pullulan; polyaminoacids and derivatives thereof,e.g., polyglutamic acids, polylysines, polyaspartic acids,polyaspartamides; maleic anhydride copolymers such as: styrene maleicanhydride copolymer, divinylethyl ether maleic anhydride copolymer;polyvinyl alcohols; copolymers thereof; terpolymers thereof; mixturesthereof; and derivatives of the foregoing.

The proportion of polyethylene glycol molecules to protein moleculeswill vary, as will their concentrations in the reaction mixture. Ingeneral, the optimum ratio (in terms of efficiency of reaction in thatthere is minimal excess unreacted protein or polymer) may be determinedby the molecular weight of the polyethylene glycol selected and on thenumber of available reactive groups available. As relates to molecularweight, typically the higher the molecular weight of the polymer, thefewer number of polymer molecules which may be attached to the protein.Similarly, branching of the polymer should be taken into account whenoptimizing these parameters. Generally, the higher the molecular weight(or the more branches) the higher the polymer:protein ratio.

As used herein, and when contemplating PEG:hGH polypeptide conjugates,the term “therapeutically effective amount” refers to an amount whichgives the desired benefit to a patient. The amount will vary from oneindividual to another and will depend upon a number of factors,including the overall physical condition of the patient and theunderlying cause of the condition to be treated. The amount of hGHpolypeptide used for therapy gives an acceptable rate of change andmaintains desired response at a beneficial level. A therapeuticallyeffective amount of the present compositions may be readily ascertainedby one of ordinary skill in the art using publicly available materialsand procedures.

The water soluble polymer may be any structural form including but notlimited to linear, forked or branched. Typically, the water solublepolymer is a poly(alkylene glycol), such as poly(ethylene glycol) (PEG),but other water soluble polymers can also be employed. By way ofexample, PEG is used to describe certain embodiments of this invention.

PEG is a well-known, water soluble polymer that is commerciallyavailable or can be prepared by ring-opening polymerization of ethyleneglycol according to methods known to those of ordinary skill in the art(Sandler and Karo, Polymer Synthesis, Academic Press, New York, Vol. 3,pages 138-161). The term “PEG” is used broadly to encompass anypolyethylene glycol molecule, without regard to size or to modificationat an end of the PEG, and can be represented as linked to the hGHpolypeptide by the formula:XO—(CH₂CH₂O)_(n)—CH₂CH₂—Ywhere n is 2 to 10,000 and X is H or a terminal modification, includingbut not limited to, a C₁₋₄ alkyl, a protecting group, or a terminalmodification group.

In some cases, a PEG used in the invention terminates on one end withhydroxy or methoxy, i.e., X is H or CH₃ (“methoxy PEG”). Alternatively,the PEG can terminate with a reactive group, thereby forming abifunctional polymer. Typical reactive groups can include those reactivegroups that are commonly used to react with the functional groups foundin the 20 common amino acids (including but not limited to, maleimidegroups, activated carbonates (including but not limited to,p-nitrophenyl ester), activated esters (including but not limited to,N-hydroxysuccinimide, p-nitrophenyl ester) and aldehydes) as well asfunctional groups that are inert to the 20 common amino acids but thatreact specifically with complementary functional groups present innon-naturally encoded amino acids. It is noted that the other end of thePEG, which is shown in the above formula by Y, will attach eitherdirectly or indirectly to a hGH polypeptide via a naturally-occurring ornon-naturally encoded amino acid. In some embodiments, a strongnucleophile (including but not limited to, hydrazine, hydrazide,hydroxylamine, semicarbazide) can be reacted with an aldehyde or ketonegroup present in a non-naturally encoded amino acid to form a hydrazone,oxime or semicarbazone, as applicable, which in some cases can befurther reduced by treatment with an appropriate reducing agent.Alternatively, the strong nucleophile can be incorporated into the hGHpolypeptide via a non-naturally encoded amino acid and used to reactpreferentially with a ketone or aldehyde group present in the watersoluble polymer.

Any molecular mass for a PEG can be used as practically desired,including but not limited to, from about 100 Daltons (Da) to 100,000 Daor more as desired (including but not limited to, sometimes 0.1-50 kDaor 10-40 kDa). The molecular weight of PEG may be of a wide range,including but not limited to, between about 100 Da and about 100,000 Daor more. The molecular weight of PEG may be between about 100 Da andabout 100,000 Da, including but not limited to, 100,000 Da, 95,000 Da,90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da,25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, 1,000 Da, 900 Da,800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, and 100 Da. Insome embodiments, the molecular weight of PEG is between about 100 Daand 50,000 Da. In some embodiments, the molecular weight of PEG isbetween about 100 Da and 40,000 Da. In some embodiments, the molecularweight of PEG is between about 1,000 Da and 40,000 Da. In someembodiments, the molecular weight of PEG is between about 5,000 Da and40,000 Da. In some embodiments, the molecular weight of PEG is betweenabout 10,000 Da and 40,000 Da. Branched chain PEGs, including but notlimited to, PEG molecules with each chain having a MW ranging from 1-100kDa (including but not limited to, 1-50 kDa or 5-20 kDa) can also beused. The molecular weight of the branched chain PEG may be, includingbut not limited to, between about 1,000 Da and about 100,000 Da or more.The molecular weight of the branched chain PEG may be between about1,000 Da and about 100,000 Da, including but not limited to, 100,000 Da,95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da,30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, and1,000 Da. In some embodiments, the molecular weight of the branchedchain PEG is between about 1,000 Da and 50,000 Da. In some embodiments,the molecular weight of the branched chain PEG is between about 1,000 Daand 40,000 Da. In some embodiments, the molecular weight of the branchedchain PEG is between about 5,000 Da and 40,000 Da. In some embodiments,the molecular weight of the branched chain PEG is between about 5,000 Daand 20,000 Da. A wide range of PEG molecules are described in, includingbut not limited to, the Shearwater Polymers, Inc. catalog, NektarTherapeutics catalog, incorporated herein by reference.

Generally, at least one terminus of the PEG molecule is available forreaction with the non-naturally-encoded amino acid. In some embodiments,the hGH polypeptide variant with a PEG derivative contains a chemicalfunctionality that is reactive with the chemical functionality presenton the side chain of the non-naturally encoded amino acid.

The polymer backbone of the water-soluble polymer can be poly(ethyleneglycol). However, it should be understood that a wide variety of watersoluble polymers including but not limited to poly(ethylene)glycol andother related polymers, including poly(dextran) and poly(propyleneglycol), are also suitable for use in the practice of this invention andthat the use of the term PEG or poly(ethylene glycol) is intended toencompass and include all such molecules. The term PEG includes, but isnot limited to, poly(ethylene glycol) in any of its forms, includingbifunctional PEG, multiarmed PEG, derivatized PEG, forked PEG, branchedPEG, pendent PEG (i.e. PEG or related polymers having one or morefunctional groups pendent to the polymer backbone), or PEG withdegradable linkages therein.

PEG is typically clear, colorless, odorless, soluble in water, stable toheat, inert to many chemical agents, does not hydrolyze or deteriorate,and is generally non-toxic. Poly(ethylene glycol) is considered to bebiocompatible, which is to say that PEG is capable of coexistence withliving tissues or organisms without causing harm. More specifically, PEGis substantially non-immunogenic, which is to say that PEG does not tendto produce an immune response in the body. When attached to a moleculehaving some desirable function in the body, such as a biologicallyactive agent, the PEG tends to mask the agent and can reduce oreliminate any immune response so that an organism can tolerate thepresence of the agent. PEG conjugates tend not to produce a substantialimmune response or cause clotting or other undesirable effects. PEGhaving the formula —CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—, where n is from about3 to about 4000, typically from about 20 to about 2000, is suitable foruse in the present invention. PEG having a molecular weight of fromabout 800 Da to about 100,000 Da are in some embodiments of the presentinvention particularly useful as the polymer backbone. The molecularweight of PEG may be of a wide range, including but not limited to,between about 100 Da and about 100,000 Da or more. The molecular weightof PEG may be between about 100 Da and about 100,000 Da, including butnot limited to, 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da,75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da,10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da,3,000 Da, 2,000 Da, 1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da,400 Da, 300 Da, 200 Da, and 100 Da. In some embodiments, the molecularweight of PEG is between about 100 Da and 50,000 Da. In someembodiments, the molecular weight of PEG is between about 100 Da and40,000 Da. In some embodiments, the molecular weight of PEG is betweenabout 1,000 Da and 40,000 Da. In some embodiments, the molecular weightof PEG is between about 5,000 Da and 40,000 Da. In some embodiments, themolecular weight of PEG is between about 10,000 Da and 40,000 Da.

The polymer backbone can be linear or branched. Branched polymerbackbones are generally known in the art. Typically, a branched polymerhas a central branch core moiety and a plurality of linear polymerchains linked to the central branch core. PEG is commonly used inbranched forms that can be prepared by addition of ethylene oxide tovarious polyols, such as glycerol, glycerol oligomers, pentaerythritoland sorbitol. The central branch moiety can also be derived from severalamino acids, such as lysine. The branched poly(ethylene glycol) can berepresented in general form as R(—PEG-OH)_(m) in which R is derived froma core moiety, such as glycerol, glycerol oligomers, or pentaerythritol,and m represents the number of arms. Multi-armed PEG molecules, such asthose described in U.S. Pat. Nos. 5,932,462 5,643,575; 5,229,490;4,289,872; U.S. Pat. Appl. 2003/0143596; WO 96/21469; and WO 93/21259,each of which is incorporated by reference herein in its entirety, canalso be used as the polymer backbone.

Branched PEG can also be in the form of a forked PEG represented byPEG(—YCHZ₂)_(n), where Y is a linking group and Z is an activatedterminal group linked to CH by a chain of atoms of defined length.

Yet another branched form, the pendant PEG, has reactive groups, such ascarboxyl, along the PEG backbone rather than at the end of PEG chains.

In addition to these forms of PEG, the polymer can also be prepared withweak or degradable linkages in the backbone. For example, PEG can beprepared with ester linkages in the polymer backbone that are subject tohydrolysis. As shown below, this hydrolysis results in cleavage of thepolymer into fragments of lower molecular weight:-PEG-CO₂-PEG-+H₂O→PEG-CO₂H+HO-PEG-It is understood by those of ordinary skill in the art that the termpoly(ethylene glycol) or PEG represents or includes all the forms knownin the art including but not limited to those disclosed herein.

Many other polymers are also suitable for use in the present invention.In some embodiments, polymer backbones that are water-soluble, with from2 to about 300 termini, are particularly useful in the invention.Examples of suitable polymers include, but are not limited to, otherpoly(alkylene glycols), such as poly(propylene glycol) (“PPG”),copolymers thereof (including but not limited to copolymers of ethyleneglycol and propylene glycol), terpolymers thereof, mixtures thereof, andthe like. Although the molecular weight of each chain of the polymerbackbone can vary, it is typically in the range of from about 800 Da toabout 100,000 Da, often from about 6,000 Da to about 80,000 Da. Themolecular weight of each chain of the polymer backbone may be betweenabout 100 Da and about 100,000 Da, including but not limited to, 100,000Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da,65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da,8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da,1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200Da, and 100 Da. In some embodiments, the molecular weight of each chainof the polymer backbone is between about 100 Da and 50,000 Da. In someembodiments, the molecular weight of each chain of the polymer backboneis between about 100 Da and 40,000 Da. In some embodiments, themolecular weight of each chain of the polymer backbone is between about1,000 Da and 40,000 Da. In some embodiments, the molecular weight ofeach chain of the polymer backbone is between about 5,000 Da and 40,000Da. In some embodiments, the molecular weight of each chain of thepolymer backbone is between about 10,000 Da and 40,000 Da.

Those of ordinary skill in the art will recognize that the foregoinglist for substantially water soluble backbones is by no means exhaustiveand is merely illustrative, and that all polymeric materials having thequalities described above are contemplated as being suitable for use inthe present invention.

In some embodiments of the present invention the polymer derivatives are“multi-functional”, meaning that the polymer backbone has at least twotermini, and possibly as many as about 300 termini, functionalized oractivated with a functional group. Multifunctional polymer derivativesinclude, but are not limited to, linear polymers having two termini,each terminus being bonded to a functional group which may be the sameor different.

Water soluble polymers can be linked to the hGH polypeptides of theinvention. The water soluble polymers may be linked via a non-naturallyencoded amino acid incorporated in the hGH polypeptide or any functionalgroup or substituent of a non-naturally encoded or naturally encodedamino acid, or any functional group or substituent added to anon-naturally encoded or naturally encoded amino acid. Alternatively,the water soluble polymers are linked to a hGH polypeptide incorporatinga non-naturally encoded amino acid via a naturally-occurring amino acid(including but not limited to, cysteine, lysine or the amine group ofthe N-terminal residue). In some cases, the hGH polypeptides of theinvention comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 non-natural aminoacids, wherein one or more non-naturally-encoded amino acid(s) arelinked to water soluble polymer(s) (including but not limited to, PEGand/or oligosaccharides). In some cases, the hGH polypeptides of theinvention further comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or morenaturally-encoded amino acid(s) linked to water soluble polymers. Insome cases, the hGH polypeptides of the invention comprise one or morenon-naturally encoded amino acid(s) linked to water soluble polymers andone or more naturally-occurring amino acids linked to water solublepolymers. In some embodiments, the water soluble polymers used in thepresent invention enhance the serum half-life of the hGH polypeptiderelative to the unconjugated form.

The number of water soluble polymers linked to a hGH polypeptide (i.e.,the extent of PEGylation or glycosylation) of the present invention canbe adjusted to provide an altered (including but not limited to,increased or decreased) pharmacologic, pharmacokinetic orpharmacodynamic characteristic such as in vivo half-life. In someembodiments, the half-life of hGH is increased at least about 10, 20,30, 40, 50, 60, 70, 80, 90 percent, 2-fold, 5-fold, 10-fold, 50-fold, orat least about 100-fold over an unmodified polypeptide.

The degree and sites at which the water soluble polymer(s) are linked tothe hGH polypeptide can modulate the binding of the hGH polypeptide tothe hGH polypeptide receptor at Site 1 or binding partner. In someembodiments, the linkages are arranged such that the hGH polypeptidebinds the hGH polypeptide receptor at Site 1 with a K_(d) of about 400nM or lower, with a K_(d) of 150 nM or lower, and in some cases with aK_(d) of 100 nM or lower, as measured by an equilibrium binding assay,such as that described in Spencer et al., J. Biol. Chem., 263:7862-7867(1988) for hGH.

Methods and chemistry for activation of polymers as well as forconjugation of peptides are described in the literature and are known inthe art. Commonly used methods for activation of polymers include, butare not limited to, activation of functional groups with cyanogenbromide, periodate, glutaraldehyde, biepoxides, epichlorohydrin,divinylsulfone, carbodiimide, sulfonyl halides, trichlorotriazine, etc.(see, R. F. Taylor, (1991), PROTEIN IMMOBILISATION FUNDAMENTAL ANDAPPLICATIONS, Marcel Dekker, N.Y.; S. S. Wong, (1992), CHEMISTRY OFPROTEIN CONJUGATION AND CROSSLINKING, CRC Press, Boca Raton; G. T.Hermanson et al., (1993), IMMOBILIZED AFFINITY LIGAND TECHNIQUES,Academic Press, N.Y.; Dunn, R. L., et al., Eds. POLYMERIC DRUGS AND DRUGDELIVERY SYSTEMS, ACS Symposium Series Vol. 469, American ChemicalSociety, Washington, D.C. 1991).

Several reviews and monographs on the functionalization and conjugationof PEG are available. See, for example, Harris, Macromol. Chem. Phys.C25: 325-373 (1985); Scouten, Methods in Enzymology 135: 30-65 (1987);Wong et al., Enzyme Microb. Technol. 14: 866-874 (1992); Delgado et al.,Critical Reviews in Therapeutic Drug Carrier Systems 9: 249-304 (1992);Zalipsky, Bioconjugate Chem. 6: 150-165 (1995).

Methods for activation of polymers can also be found in WO 94/17039,U.S. Pat. No. 5,324,844, WO 94/18247, WO 94/04193, U.S. Pat. No.5,219,564, U.S. Pat. No. 5,122,614, WO 90/13540, U.S. Pat. No.5,281,698, and WO 93/15189, and for conjugation between activatedpolymers and enzymes including but not limited to Coagulation FactorVIII (WO 94/15625), hemoglobin (WO 94/09027), oxygen carrying molecule(U.S. Pat. No. 4,412,989), ribonuclease and superoxide dismutase(Veronese at al., App. Biochem. Biotech. 11: 141-52 (1985)). Allreferences and patents cited are incorporated by reference herein.

The reaction products are subsequently subjected to hydrophobicinteraction chromatography to separate the PEGylated hGH polypeptidevariants from free PEG. Suitable conditions vary depending on therelative sizes of the cross-linked complexes versus the desiredconjugates and are readily determined by those of ordinary skill in theart. The eluent containing the desired conjugates may be concentrated byultrafiltration and desalted by diafiltration.

If necessary, the PEGylated hGH polypeptide obtained from thehydrophobic chromatography can be purified further by one or moreprocedures known to those of ordinary skill in the art including, butare not limited to, affinity chromatography; anion- or cation-exchangechromatography (using, including but not limited to, DEAE SEPHAROSE);chromatography on silica; reverse phase HPLC; gel filtration (using,including but not limited to, SEPHADEX G-75); hydrophobic interactionchromatography; size-exclusion chromatography, metal-chelatechromatography; ultrafiltration/diafiltration; ethanol precipitation;ammonium sulfate precipitation; chromatofocusing; displacementchromatography; electrophoretic procedures (including but not limited topreparative isoelectric focusing), differential solubility (includingbut not limited to ammonium sulfate precipitation), or extraction.Apparent molecular weight may be estimated by GPC by comparison toglobular protein standards (Preneta, Ariz. in PROTEIN PURIFICATIONMETHODS, A PRACTICAL APPROACH (Harris & Angal, Eds.) IRL Press 1989,293-306). The purity of the hGH-PEG conjugate can be assessed byproteolytic degradation (including but not limited to, trypsin cleavage)followed by mass spectrometry analysis. Pepinsky RB., et al., J.Pharmcol. & Exp. Ther. 297(3):1059-66 (2001).

A water soluble polymer linked to an amino acid of a hGH polypeptide ofthe invention can be further derivatized or substituted withoutlimitation.

Other PEG Derivatives and General PEGylation Techniques

Other exemplary PEG molecules that may be linked to hGH polypeptides, aswell as PEGylation methods include those described in, e.g., U.S. PatentPublication No. 2004/0001838; 2002/0052009; 2003/0162949; 2004/0013637;2003/0228274; 2003/0220447; 2003/0158333; 2003/0143596; 2003/0114647;2003/0105275; 2003/0105224; 2003/0023023; 2002/0156047; 2002/0099133;2002/0086939; 2002/0082345; 2002/0072573; 2002/0052430; 2002/0040076;2002/0037949; 2002/0002250; 2001/0056171; 2001/0044526; 2001/0021763;U.S. Pat. Nos. 6,646,110; 5,824,778; 5,476,653; 5,219,564; 5,629,384;5,736,625; 4,902,502; 5,281,698; 5,122,614; 5,473,034; 5,516,673;5,382,657; 6,552,167; 6,610,281; 6,515,100; 6,461,603; 6,436,386;6,214,966; 5,990,237; 5,900,461; 5,739,208; 5,672,662; 5,446,090;5,808,096; 5,612,460; 5,324,844; 5,252,714; 6,420,339; 6,201,072;6,451,346; 6,306,821; 5,559,213; 5,747,646; 5,834,594; 5,849,860;5,980,948; 6,004,573; 6,129,912; WO 97/32607, EP 229,108, EP 402,378, WO92/16555, WO 94/04193, WO 94/14758, WO 94/17039, WO 94/18247, WO94/28024, WO 95/00162, WO 95/11924, WO95/13090, WO 95/33490, WO96/00080, WO 97/18832, WO 98/41562, WO 98/48837, WO 99/32134, WO99/32139, WO 99/32140, WO 96/40791, WO 98/32466, WO 95/06058, EP439.508, WO 97/03106, WO 96/21469, WO 95/13312, EP 921 131, WO 98/05363,EP 809 996, WO 96/41813, WO 96/07670, EP 605 963, EP 510 356, EP 400472, EP 183 503 and EP 154 316, which are incorporated by referenceherein. Any of the PEG molecules described herein may be used in anyform, including but not limited to, single chain, branched chain,multiarm chain, single functional, bi-functional, multi-functional, orany combination thereof.

Enhancing Affinity for Serum Albumin

Various molecules can also be fused to the hGH polypeptides of theinvention to modulate the half-life of hGH polypeptides in serum. Insome embodiments, molecules are linked or fused to hGH polypeptides ofthe invention to enhance affinity for endogenous serum albumin in ananimal.

For example, in some cases, a recombinant fusion of a hGH polypeptideand an albumin binding sequence is made. Exemplary albumin bindingsequences include, but are not limited to, the albumin binding domainfrom streptococcal protein G (see. e g., Makrides et al., J. Pharmacol.Exp. Ther. 277:534-542 (1996) and Sjolander et al., J, Immunol. Methods201:115-123 (1997)), or albumin-binding peptides such as those describedin, e.g., Dennis, et al., J. Biol. Chem. 277:35035-35043 (2002).

In other embodiments, the hGH polypeptides of the present invention areacylated with fatty acids. In some cases, the fatty acids promotebinding to serum albumin. See, e.g., Kurtzhals, et al., Biochem. J.312:725-731 (1995).

In other embodiments, the hGH polypeptides of the invention are fuseddirectly with serum albumin (including but not limited to, human serumalbumin). Those of skill in the art will recognize that a wide varietyof other molecules can also be linked to hGH in the present invention tomodulate binding to serum albumin or other serum components.

XVI. Glycosylation of hGH Polypeptides

The invention includes hGH polypeptides incorporating one or morenon-naturally encoded amino acids bearing saccharide residues. Thesaccharide residues may be either natural (including but not limited to,N-acetylglucosamine) or non-natural (including but not limited to,3-fluorogalactose). The saccharides may be linked to the non-naturallyencoded amino acids either by an N- or O-linked glycosidic linkage(including but not limited to, N-acetylgalactose-L-serine) or anon-natural linkage (including but not limited to, an oxime or thecorresponding C- or S-linked glycoside).

The saccharide (including but not limited to, glycosyl) moieties can beadded to hGH polypeptides either in vivo or in vitro. In someembodiments of the invention, a hGH polypeptide comprising acarbonyl-containing non-naturally encoded amino acid is modified with asaccharide derivatized with an aminooxy group to generate thecorresponding glycosylated polypeptide linked via an oxime linkage. Onceattached to the non-naturally encoded amino acid, the saccharide may befurther elaborated by treatment with glycosyltransferases and otherenzymes to generate an oligosaccharide bound to the hGH polypeptide.See, e.g., H. Liu, et al. J. Am. Chem. Soc. 125: 1702-1703 (2003).

XVII. GH Supergene Family Member Dimers and Multimers

The present invention also provides for GH supergene family membercombinations (including but not limited to hGH and hGH analogs) such ashomodimers, heterodimers, homomultimers, or heteromultimers (i.e.,trimers, tetramers, etc.) where a GH supergene family member polypeptidesuch as hGH containing one or more non-naturally encoded amino acids isbound to another GH supergene family member or variant thereof or anyother polypeptide that is a non-GH supergene family member or variantthereof, either directly to the polypeptide backbone or via a linker.Due to its increased molecular weight compared to monomers, the GHsupergene family member, such as hGH, dimer or multimer conjugates mayexhibit new or desirable properties, including but not limited todifferent pharmacological, pharmacokinetic, pharmacodynamic, modulatedtherapeutic half-life, or modulated plasma half-life relative to themonomeric GH supergene family member. In some embodiments, the GHsupergene family member, such as hGH, dimers of the invention willmodulate the dimerization of the GH supergene family member receptor. Inother embodiments, the GH supergene family member dimers or multimers ofthe present invention will act as a GH supergene family member receptorantagonist, agonist, or modulator.

In some embodiments, one or more of the hGH molecules present in a hGHcontaining dimer or multimer comprises a non-naturally encoded aminoacid linked to a water soluble polymer that is present within the SiteII binding region. As such, each of the hGH molecules of the dimer ormultimer are accessible for binding to the hGH polypeptide receptor viathe Site I interface but are unavailable for binding to a second hGHpolypeptide receptor via the Site II interface. Thus, the hGHpolypeptide dimer or multimer can engage the Site I binding sites ofeach of two distinct hGH polypeptide receptors but, as the hGH moleculeshave a water soluble polymer attached to a non-genetically encoded aminoacid present in the Site II region, the hGH polypeptide receptors cannotengage the Site II region of the hGH polypeptide ligand and the dimer ormultimer acts as a hGH polypeptide antagonist. In some embodiments, oneor more of the hGH molecules present in a hGH polypeptide containingdimer or multimer comprises a non-naturally encoded amino acid linked toa water soluble polymer that is present within the Site I bindingregion, allowing binding to the Site II region. Alternatively, in someembodiments one or more of the hGH molecules present in a hGHpolypeptide containing dimer or multimer comprises a non-naturallyencoded amino acid linked to a water soluble polymer that is present ata site that is not within the Site I or Site II binding region, suchthat both are available for binding. In some embodiments a combinationof hGH molecules is used having Site I, Site II, or both available forbinding. A combination of hGH molecules wherein at least one has Site Iavailable for binding, and at least one has Site II available forbinding may provide molecules having a desired activity or property. Inaddition, a combination of hGH molecules having both Site I and Site IIavailable for binding may produce a super-agonist hGH molecule.

In some embodiments, the GH supergene family member polypeptides arelinked directly, including but not limited to, via an Asn-Lys amidelinkage or Cys-Cys disulfide linkage. In some embodiments, the linked GHsupergene family member polypeptides, and/or the linked non-GH supergenefamily member, will comprise different non-naturally encoded amino acidsto facilitate dimerization.

Alternatively, the two GH supergene family member polypeptides, and/orthe linked non-GH supergene family member, are linked via a linker. Anyhetero- or homo-bifunctional linker can be used to link the two GHsupergene family members, and/or the linked non-GH supergene familymember, polypeptides, which can have the same or different primarysequence. In some cases, the linker used to tether the GH supergenefamily member, and/or the linked non-GH supergene family member,polypeptides together can be a bifunctional PEG reagent. The linker mayhave a wide range of molecular weight or molecular length. Larger orsmaller molecular weight linkers may be used to provide a desiredspatial relationship or conformation between the hGH and the linkedentity.

In some embodiments, the invention provides water-soluble bifunctionallinkers that have a dumbbell structure that includes: a) a firstfunctional group on at least a first end of a polymer backbone; and b)at least a second functional group on a second end of the polymerbackbone. The second functional group can be the same or different asthe first functional group. The second functional group, in someembodiments, is not reactive with the first functional group. Theinvention provides, in some embodiments, water-soluble compounds thatcomprise at least one arm of a branched molecular structure. Forexample, the branched molecular structure can be dendritic. In someembodiments, the invention provides multimers comprising one or more GHsupergene family member, such as hGH, formed by reactions with watersoluble activated polymers.

XVIII. Administration and Pharmaceutical Compositions

The polypeptides or proteins of the invention (including but not limitedto, hGH, synthetases, proteins comprising one or more unnatural aminoacid, etc.) are optionally employed for therapeutic uses, including butnot limited to, in combination with a suitable pharmaceutical carrier.Such compositions, for example, comprise a therapeutically effectiveamount of the compound, and a pharmaceutically acceptable carrier orexcipient. Such a carrier or excipient includes, but is not limited to,saline, buffered saline, dextrose, water, glycerol, ethanol, and/orcombinations thereof. The formulation is made to suit the mode ofadministration. In general, methods of administering proteins are knownto those of ordinary skill in the art and can be applied toadministration of the polypeptides of the invention.

Therapeutic compositions comprising one or more polypeptide of theinvention are optionally tested in one or more appropriate in vitroand/or in vivo animal models of disease, to confirm efficacy, tissuemetabolism, and to estimate dosages, according to methods known to thoseof ordinary skill in the art. In particular, dosages can be initiallydetermined by activity, stability or other suitable measures ofunnatural herein to natural amino acid homologues (including but notlimited to, comparison of a hGH polypeptide modified to include one ormore unnatural amino acids to a natural amino acid hGH polypeptide),i.e., in a relevant assay.

Administration is by any of the routes normally used for introducing amolecule into ultimate contact with blood or tissue cells. The unnaturalamino acid polypeptides of the invention are administered in anysuitable manner, optionally with one or more pharmaceutically acceptablecarriers. Suitable methods of administering such polypeptides in thecontext of the present invention to a patient are available, and,although more than one route can be used to administer a particularcomposition, a particular route can often provide a more immediate andmore effective action or reaction than another route.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositions of thepresent invention.

hGH polypeptides of the invention may be administered by anyconventional route suitable for proteins or peptides, including, but notlimited to parenterally, e.g. injections including, but not limited to,subcutaneously or intravenously or any other form of injections orinfusions. Polypeptide compositions can be administered by a number ofroutes including, but not limited to oral, intravenous, intraperitoneal,intramuscular, transdermal, subcutaneous, topical, sublingual, or rectalmeans. Compositions comprising non-natural amino acid polypeptides,modified or unmodified, can also be administered via liposomes. Suchadministration routes and appropriate formulations are generally knownto those of skill in the art. The hGH polypeptide comprising anon-naturally encoded amino acid, may be used alone or in combinationwith other suitable components such as a pharmaceutical carrier.

The hGH polypeptide comprising a non-natural amino acid, alone or incombination with other suitable components, can also be made intoaerosol formulations (i.e., they can be “nebulized”) to be administeredvia inhalation. Aerosol formulations can be placed into pressurizedacceptable propellants, such as dichlorodifluoromethane, propane,nitrogen, and the like.

Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intradermal, intraperitoneal, and subcutaneous routes, include aqueousand non-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The formulations of hGH can be presented in unit-dose or multi-dosesealed containers, such as ampules and vials.

Parenteral administration and intravenous administration are preferredmethods of administration. In particular, the routes of administrationalready in use for natural amino acid homologue therapeutics (includingbut not limited to, those typically used for EPO, GH, G-CSF, GM-CSF,IFNs, interleukins, antibodies, and/or any other pharmaceuticallydelivered protein), along with formulations in current use, providepreferred routes of administration and formulation for the polypeptidesof the invention.

The dose administered to a patient, in the context of the presentinvention, is sufficient to have a beneficial therapeutic response inthe patient over time, or other appropriate activity, depending on theapplication. The dose is determined by the efficacy of the particularvector, or formulation, and the activity, stability or serum half-lifeof the unnatural amino acid polypeptide employed and the condition ofthe patient, as well as the body weight or surface area of the patientto be treated. The size of the dose is also determined by the existence,nature, and extent of any adverse side-effects that accompany theadministration of a particular vector, formulation, or the like in aparticular patient.

In determining the effective amount of the vector or formulation to beadministered in the treatment or prophylaxis of disease (including butnot limited to, cancers, inherited diseases, diabetes, AIDS, or thelike), the physician evaluates circulating plasma levels, formulationtoxicities, progression of the disease, and/or where relevant, theproduction of anti-unnatural amino acid polypeptide antibodies.

The dose administered, for example, to a 70 kilogram patient, istypically in the range equivalent to dosages of currently-usedtherapeutic proteins, adjusted for the altered activity or serumhalf-life of the relevant composition. The vectors or pharmaceuticalformulations of this invention can supplement treatment conditions byany known conventional therapy, including antibody administration,vaccine administration, administration of cytotoxic agents, naturalamino acid polypeptides, nucleic acids, nucleotide analogues, biologicresponse modifiers, and the like.

For administration, formulations of the present invention areadministered at a rate determined by the LD-50 or ED-50 of the relevantformulation, and/or observation of any side-effects of the unnaturalamino acid polypeptides at various concentrations, including but notlimited to, as applied to the mass and overall health of the patient.Administration can be accomplished via single or divided doses.

If a patient undergoing infusion of a formulation develops fevers,chills, or muscle aches, he/she receives the appropriate dose ofaspirin, ibuprofen, acetaminophen or other pain/fever controlling drug.Patients who experience reactions to the infusion such as fever, muscleaches, and chills are premedicated 30 minutes prior to the futureinfusions with either aspirin, acetaminophen, or, including but notlimited to, diphenhydramine. Meperidine is used for more severe chillsand muscle aches that do not quickly respond to antipyretics andantihistamines. Cell infusion is slowed or discontinued depending uponthe severity of the reaction.

Human hGH polypeptides of the invention can be administered directly toa mammalian subject. Administration is by any of the routes normallyused for introducing hGH polypeptide to a subject. The hGH polypeptidecompositions according to embodiments of the present invention includethose suitable for oral, rectal, topical, inhalation (including but notlimited to, via an aerosol), buccal (including but not limited to,sub-lingual), vaginal, parenteral (including but not limited to,subcutaneous, intramuscular, intradermal, intraarticular, intrapleural,intraperitoneal, inracerebral, intraarterial, or intravenous), topical(i.e., both skin and mucosal surfaces, including airway surfaces) andtransdermal administration, although the most suitable route in anygiven case will depend on the nature and severity of the condition beingtreated. Administration can be either local or systemic. Theformulations of compounds can be presented in unit-dose or multi-dosesealed containers, such as ampoules and vials. hGH polypeptides of theinvention can be prepared in a mixture in a unit dosage injectable form(including but not limited to, solution, suspension, or emulsion) with apharmaceutically acceptable carrier. hGH polypeptides of the inventioncan also be administered by continuous infusion (using, including butnot limited to, minipumps such as osmotic pumps), single bolus orslow-release depot formulations.

Formulations suitable for administration include aqueous and non-aqueoussolutions, isotonic sterile solutions, which can contain antioxidants,buffers, bacteriostats, and solutes that render the formulationisotonic, and aqueous and non-aqueous sterile suspensions that caninclude suspending agents, solubilizers, thickening agents, stabilizers,and preservatives. Solutions and suspensions can be prepared fromsterile powders, granules, and tablets of the kind previously described.

Freeze-drying is a commonly employed technique for presenting proteinswhich serves to remove water from the protein preparation of interest.Freeze-drying, or lyophilization, is a process by which the material tobe dried is first frozen and then the ice or frozen solvent is removedby sublimation in a vacuum environment. An excipient may be included inpre-lyophilized formulations to enhance stability during thefreeze-drying process and/or to improve stability of the lyophilizedproduct upon storage. Pikal, M. Biopharm. 3(9)26-30 (1990) and Arakawaet al. Pharm. Res. 8(3):285-291 (1991).

The spray drying of pharmaceuticals is also known to those of ordinaryskill in the art. For example, see Broadhead, J. et al., “The SprayDrying of Pharmaceuticals,” in Drug Dev. Ind. Pharm, 18 (11 & 12),1169-1206 (1992). In addition to small molecule pharmaceuticals, avariety of biological materials have been spray dried and these include:enzymes, sera, plasma, micro-organisms and yeasts. Spray drying is auseful technique because it can convert a liquid pharmaceuticalpreparation into a fine, dustless or agglomerated powder in a one-stepprocess. The basic technique comprises the following four steps: a)atomization of the feed solution into a spray; b) spray-air contact; c)drying of the spray; and d) separation of the dried product from thedrying air. U.S. Pat. Nos. 6,235,710 and 6,001,800, which areincorporated by reference herein, describe the preparation ofrecombinant erythropoietin by spray drying.

The pharmaceutical compositions and formulations of the invention maycomprise a pharmaceutically acceptable carrier, excipient, orstabilizer. Pharmaceutically acceptable carriers are determined in partby the particular composition being administered, as well as by theparticular method used to administer the composition. Accordingly, thereis a wide variety of suitable formulations of pharmaceuticalcompositions (including optional pharmaceutically acceptable carriers,excipients, or stabilizers) of the present invention (see, e.g.,Remington's Pharmaceutical Sciences, 17^(th) ed. 1985)).

Suitable carriers include buffers containing succinate, phosphate,borate, HEPES, citrate, histidine or histidine derivatives, imidazole,acetate, bicarbonate, and other organic acids; antioxidants includingbut not limited to, ascorbic acid; low molecular weight polypeptidesincluding but not limited to those less than about 10 residues;proteins, including but not limited to, serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers including but not limited to,polyvinylpyrrolidone; amino acids including but not limited to, glycine,glutamine, asparagine, arginine, histidine or histidine derivatives,methionine, glutamate, or lysine; monosaccharides, disaccharides, andother carbohydrates, including but not limited to, trehalose, sucrose,glucose, mannose, or dextrins; chelating agents including but notlimited to, EDTA; divalent metal ions including but not limited to,zinc, cobalt, or copper; sugar alcohols including but not limited to,mannitol or sorbitol; salt-forming counter ions including but notlimited to, sodium; and/or nonionic surfactants including but notlimited to, TWEEN™ (including but not limited to, Tween 80 (polysorbate80) and Tween 20 (polysorbate 20), PLURONICS® and other pluronic acids,including but not limited to, and other pluronic acids, including butnot limited to, pluronic acid F68 (poloxamer 188), or PEG. Suitablesurfactants include for example but are not limited to polyethers basedupon poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide),i.e., (PEO-PPO-PEO), or poly(propylene oxide)-poly(ethyleneoxide)-poly(propylene oxide), i.e., (PPO-PEO-PPO), or a combinationthereof. PEO-PPO-PEO and PPO-PEO-PPO are commercially available underthe trade names PLURONICS®, R-PLURONICS™, TETRONICS® and R-TETRONICS™(BASF Wyandotte Corp., Wyandotte, Mich.) and are further described inU.S. Pat. No. 4,820,352 incorporated herein in its entirety byreference. Other ethylene/polypropylene block polymers may be suitablesurfactants. A surfactant or a combination of surfactants may be used tostabilize PEGylated hGH against one or more stresses including but notlimited to stress that results from agitation. Some of the above may bereferred to as “bulking agents.” Some may also be referred to as“tonicity modifiers.”

hGH polypeptides of the invention, including those linked to watersoluble polymers such as PEG can also be administered by or as part ofsustained-release systems. Sustained-release compositions include,including but not limited to, semi-permeable polymer matrices in theform of shaped articles, including but not limited to, films, ormicrocapsules. Sustained-release matrices include from biocompatiblematerials such as poly(2-hydroxyethyl methacrylate) (Langer et al., J.Biomed. Mater. Res., 15: 267-277 (1981); Langer, Chem. Tech., 12: 98-105(1982), ethylene vinyl acetate (Langer et al., supra) orpoly-D-(−)-3-hydroxybutyric acid (EP 133,988), polylactides (polylacticacid) (U.S. Pat. No. 3,773,919; EP 58,481), polyglycolide (polymer ofglycolic acid), polylactide co-glycolide (copolymers of lactic acid andglycolic acid) polyanhydrides, copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (Sidman et al., Biopolymers, 22, 547-556 (1983),poly(ortho)esters, polypeptides, hyaluronic acid, collagen, chondroitinsulfate, carboxylic acids, fatty acids, phospholipids, polysaccharides,nucleic acids, polyamino acids, amino acids such as phenylalanine,tyrosine, isoleucine, polynucleotides, polyvinyl propylene,polyvinylpyrrolidone and silicone. Sustained-release compositions alsoinclude a liposomally entrapped compound. Liposomes containing thecompound are prepared by methods known per se: DE 3,218,121; Eppstein etal., Proc. Natl. Acad. Sci. U.S.A., 82: 3688-3692 (1985); Hwang et al.,Proc. Natl. Acad. Sci. U.S.A., 77: 4030-4034 (1980); EP 52,322; EP36,676; U.S. Pat. No. 4,619,794; EP 143,949; U.S. Pat. No. 5,021,234;Japanese Pat. Appln. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545;and EP 102,324. All references and patents cited are incorporated byreference herein.

Liposomally entrapped hGH polypeptides can be prepared by methodsdescribed in, e.g., DE 3,218,121; Eppstein et al., Proc. Natl. Acad.Sci. U.S.A., 82: 3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci.U.S.A., 77: 4030-4034 (1980); EP 52,322; EP 36,676; U.S. Pat. No.4,619,794; EP 143,949; U.S. Pat. No. 5,021,234; Japanese Pat. Appln.83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324.Composition and size of liposomes are well known or able to be readilydetermined empirically by one of ordinary skill in the art. Someexamples of liposomes as described in, e.g., Park J W, et al., Proc.Natl. Acad. Sci. USA 92:1327-1331 (1995); Lasic D and Papahadjopoulos D(eds): MEDICAL APPLICATIONS OF LIPOSOMES (1998); Drummond D C, et al.,Liposomal drug delivery systems for cancer therapy, in Teicher B (ed):CANCER DRUG DISCOVERY AND DEVELOPMENT (2002); Park J W, et al., Clin.Cancer Res. 8:1172-1181 (2002); Nielsen U B, et al., Biochim. Biophys.Acta 1591(1-3):109-118 (2002); Mamot C, et al., Cancer Res. 63:3154-3161 (2003). All references and patents cited are incorporated byreference herein.

The dose administered to a patient in the context of the presentinvention should be sufficient to cause a beneficial response in thesubject over time. Generally, the total pharmaceutically effectiveamount of the hGH polypeptide of the present invention administeredparenterally per dose is in the range of about 0.01 μg/kg/day to about100 μg/kg, or about 0.05 mg/kg to about 1 mg/kg, of patient body weight,although this is subject to therapeutic discretion. The frequency ofdosing is also subject to therapeutic discretion, and may be morefrequent or less frequent than the commercially available hGHpolypeptide products approved for use in humans. Generally, a PEGylatedhGH polypeptide of the invention can be administered by any of theroutes of administration described above.

It is believed that one of ordinary skill in the art, using thepreceding description, may utilize the present invention to the fullestextent. The following examples are illustrative only, and not limitingof the claims or the present disclosure, in any way whatsoever.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 8 Liter Fermentation

This example describes expression methods used for hGH polypeptidescomprising a non-natural amino acid. Host cells were transformed withconstructs for orthogonal tRNA, orthogonal aminoacyl tRNA synthetase,and a polynucleotide encoding hGH polypeptide comprising a selectorcodon.

Preparation

Sterile base, 5.5 M potassium carbonate (0.5 L), was prepared andsterilized by steam or filtration. Sterile 25% v/v polyalkylenedefoamer, such as Struktol J673 (0.1 L), was prepared and sterilized bysteam. No acid was required. Concentrated feed medium (4 L, defined) wasprepared and filter sterilized into a sterile feed tank or bioprocessbag.

The fermentor was set-up. It was sterilized with 3.91 L Base Saltssolution. The fermentor was brought to the following conditions:temperature=37° C., pH=6.9, 1 VVM air. 0.092 L concentrated feed mediumwas added to the fermentor. 4 mL of 50 mg/mL kanamycin was added.

Solutions of glycerol and arabinose (an optionally yeast extract) aswell as the following reagents were prepared:

Trace metals (steam sterilized or filter sterilized) Component g/lNa₃citrate 74 FeCl₃•6H₂O 27 CoCl₂•6H₂O 2 Na₂MoO₄•2H₂O 2 ZnSO₄•7H₂O 3MnSO₄•nH₂O 2 CuCl₂•2H₂O 1.3 CaCl₂•2H₂O 1 H₃BO₃ 0.5

Vitamins (filter sterilized) Component g/l Niacin 6.1 Pantothenic acid5.4 Pyridoxine.HCl 1.4 Thiamine.HCl 1 Riboflavin 0.42 Biotin 0.06 Folicacid 0.04

Glucose (steam sterilized or filter sterilized) Component g/l l Glucose600 1.8-2

1 M MgSO₄ (steam sterilized or filter sterilized) Component g/lMgSO₄•7H₂O 246

Ammonium sulfate, 400 g/l (steam sterilized or filter sterilized)Component g/l Ammonium sulfate 400

5.5 M K₂CO₃ (steam sterilized or filter sterilized) Component g/l or l/lK₂CO₃ 760 H₂0 0.76

1M L-leucine (filter sterilized) Component g/l or l/l L-leucine 131Conc. HCl 0.1

1M L-isoleucine (filter sterilized) Component g/l or l/l L-isoleucine131 Conc. HCl 0.1

Base salts, 1X (steam sterilized or filter sterilized) Component g/l orl/l Na₂HPO₄•7H₂O 15.4 KH₂PO₄ 6.8 NH₄Cl 4

Concentrated feed Component l/l Ammonium sulfate solution 0.194 Glucosesolution 0.537 Magnesium solution 0.029 Trace metals concentratesolution 0.045 Vitamins concentrate solution 0.045 L-isoleucine 0.054L-leucine 0.096

Batch medium Component g/l or l/l Base salts solution, 1X 0.977Concentrated feed medium 0.023Process

The process performed is described as indicated in Table 2.

TABLE 2 Day Clock Time(hr) Action −2 0800 −46 2 mL starter culture wasbegun with a 1 μL glycerol stock. The culture was shaken at 37° C., 250rpm until OD₆₀₀ = 2-6. −1 0800 −22 150 μL of starter culture wastransferred to 150 mL of defined medium in a shake flask. The culturewas incubated at 28-37° C. with aeration until OD₆₀₀ = 2-5. 1 0600 0 100mL of the seed culture was transferred to the fermentor. 1 1400 8 Thefeed pump was started. The exact timing of this was dictated by when theculture depleted the batch nutrients. Approximately 2.6 L ofconcentrated feed medium was fed to the culture over 19.5 hours using apreset feed schedule. The minimum feed rate was 0.31 mL/minute, and themaximum feed rate was 6 mL/minute. If needed, the DO (dissolved oxygen)was controlled with cascade of agitation and O₂ supplementation. 2 083026.5 200 mL bolus of 80% glycerol was added to the culture whilemaintaining the feed schedule of concentrated feed. 2 0930 27.5 Theconcentrated feed was turned off. The feed was changed to a 40% glycerolsolution, and the feed line was purged. The feed was stopped. Thenon-natural amino acid pAF was added to a final concentration of 4 mM.The culture was induced with a 8 mL bolus of 20% arabinose. 2 1130 29.5The 40% glycerol feed was turned on. 2 1930 37.5 Cells were harvested.Tight wet cell densities were from 0.2-0.3 kg/L. The cell paste at wasfrozen at −80° C.

The feed schedule was as indicated in Table 3, and the fermentation feedflow rate is shown as FIG. 1. See also FIG. 2.

TABLE 3 Approximate Flow rate time (h) (ml/min) Notes 0 0 Timesindicated are post inoculation. 8 0 8 0.31 Flow rates increased linearlyfrom one set point to the next. 10 0.42 12 0.57 14 0.77 16 1.04 18 1.4020 1.90 22 2.57 24 3.47 26 4.69 27.5 6.00 Flow was turned off at 27.5hours after purging the line with 40% glycerol. 27.5 0.00 29.5 0 29.51.90 40% glycerol feed was turned on at 29.5 hours. 37.5 1.90 37.5 0Fermentation was harvested.

Modifications to this scheme have been completed at the induction step(step IV) and harvest step (step V). After the culture reached an OD₆₀₀of about 100 to about 120, a) the glycerol bolus was delivered 1.5 hoursbefore induction; b) the pAF was added and a switch to yeastextract/glycerol feed was performed 1 hour before induction; 3)arabinose was added 0 hours before induction; 4) the induction wascompleted for 8 hours.

Example 2 hGH Purification, PEGylation, and hGH-PEG Purification ProcessCytoplasmic Preparation from E. coli

1. Cell Lysis & hGH Oxidation

An 850 gram bacterial cell pellet was resuspended in 2550 ml (3 volumes)of 20 mM TRIS, pH 8.5 lysis buffer to obtain a mixture that is 25%solid. Approximately four liters of culture in fermentation broth willyield this 850 gram bacterial pellet. The mixture was stirred at roomtemperature for 30-60 minutes, and the suspension was passed through theMicrofluidizer processor twice with cooling at 15,000 psi. The lysatewas centrifuged at 13,500×g for 45 minutes in a JA10 rotor at 4° C., andthe supernatant was collected. Freshly prepared 0.1 M GSSG (FW 612.6)was added so that the molar ratio of GSSG to hGH was approximately 16.The combination was stirred to mix well, and the pH was adjusted to7.2-7.4 with 1 M NaOH. After the mixture was stirred overnight at 4° C.,it was diluted until its conductivity is 1.6-1.9 mS/cm with water. Thesample was designated as GHQFFload with the lot number.

2. Column 1—Q Sepharose FF Chromatography

The column dimension was as indicated: INdEX100/500, 100 mmI.D.×21.5cm=1688 ml. GHQFF Buffer A consisted of 10 mM Bis-TRIS, pH 6.5 with aconductivity of 0.5 mS/cm, and GHQFF Buffer B consisted of 10 mMBis-TRIS, 1 M NaCl, pH 6.5 with a conductivity of 90 mS/cm. The flowrate was 90 ml/min for processing the sample, and 40 ml/min forcleaning.

The AKTA system was depyrogenated. To depyrogenate and equilibrate theQFF column, the “QFF depy equi” program was used: the column was washedwith 2 column volumes of MilliQ water, 2 column volumes of 1 M NaOH/1MNaCl, incubated for 30 minutes, washed with 3 column volumes of GHQFFBuffer B, then equilibrated with 4 column volumes of GHQFF Buffer A.

The sample GHQFFload was loaded onto the anion exchange column. Thecolumn was washed with 5 column volumes of GHQFF Buffer A, and elutedwith 4 column volumes of 6% GHQFF Buffer B in A. The major peak wascollected. Sample collection was initiated at approximately 0.85 mS/cmand 166 mAU and was ended at approx. 220 mAU. The collected eluate wasdesignated as GHQFFpool with the lot number, and it was brownish orangein color. The pool was stored at 4° C. overnight. The average step yieldfrom 3 batches was 84.7%.

The column was washed with 2-3 column volumes of GHQFF Buffer B. 2column volumes of 1 M NaOH/1M NaCl was pumped in, and the column wasincubated for 1-6 days. If the column was not used within 6 days, it wasrinsed with 1 column volume of 1 M NaOH/1M NaCl, 3 column volumes ofBuffer B, 2 column volumes of MilliQ water, and 2.5 column volumes of20% EtOH.

An extensive cleaning of the column was done every 3-5 cycles. Followingthe 1 M NaOH/1 M NaCl incubation, the following was performed: washedupflow with 2.5 column volumes of Q Column Cleaning Buffer, incubatedfor 60-80 hours, washed with 1.5 column volumes of MilliQ water, 1column volume from 0 to 70% EtOH, 5 column volumes of 70% EtOH, 2.5column volumes of 20% EtOH. The Q Column Cleaning Buffer consisted of0.5% Triton X-100, 0.1 M acetic acid.

3. UF/DF (Ultrafiltration/Diafiltration) I

The following filter was used for this procedure: Sartorius SartoconSlice 10 K Hydrosart cassette, 1000 cm². The GHQFFpool sample wasconcentrated down to ˜450 ml (or ˜200 ml in the retentate flask). It wasthen diafiltrated with 2.7 L (6-volume) of GHCHT Buffer A which consistsof 10 mM Bis-TRIS, 1 mM MgCl₂, pH 6.3. After collecting the retentate,the system was rinsed with 300 ml of the buffer and the rinse solutionwas combined with the retentate. The retentate was centrifuged at 4,000rpm (2,862×g) for 5 minutes, and the supernatant was collected. Thesupernatant was designated as GHCHTload with the lot number. This samplewas either processed within 2 hours or was stored at 4° C. overnight.

4. Column 2—Ceramic Hydroxyapatite (CHT) Chromatography (Type I CHT, 40μm)

The column dimension was as follows: INdEX100/500, 100 mmI.D.×10.5cm=824 ml. GHCHT Buffer A consisted of 10 mM Bis-TRIS, 1 mM MgCl₂, pH6.3 with a conductivity of 0.94 mS/cm. GHCHT Buffer B consisted of 10 mMBis-TRIS, 0.5 M MgCl₂, pH 6.3 with a conductivity of 80.5 mS/cm. Theflow rate was 90 ml/min for processing, and 40 ml/min for cleaning.

The AKTA system was depyrogenated. To depyrogenate and equilibrate theCHT column, the “CHT depy equi” program was run: the CHT column waswashed with 2 column volumes of MilliQ water, 2 column volumes of 1 MNaOH/1 M NaCl, incubated for 30 minutes, washed with 3 column volumes of0.5 M NaPO₄/pH 7.0, and then equilibrated with 4 column volumes of GHCHTBuffer A. The GHCHTload sample was then loaded onto the column. Thecolumn was washed with 5 column volumes of GHCHT Buffer A.

Elution was performed with a linear gradient of 0-40% GHCHT Buffer Bover 5 column volumes, a step gradient of 40% GHCHT Buffer B over 3column volumes, and washed with 100% GHCHT Buffer B over 2 columnvolumes. The main peak was collected. The collection was started atapproximately 26 mAU, 20 mS/cm, 28% GHCHT Buffer B and was ended atapprox. 86 mAU, 34 mS/cm, 40% GHCHT Buffer B. The collected eluate wasdesignated as GHCHTpool with the lot #. The pool was stored at 4° C.overnight. The average step yield from 3 batches was 96.3%.

The CHT column was washed with 3 column volumes of 0.5 M NaPO₄/pH 7.0.The column was left in this phosphate buffer, or the following wasperformed: washed the column upflow with 2 column volumes of 1 M NaOH/1M NaCl, 3 column volumes of 0.5 M NaPO₄/pH 7.0, 2.5 column volumes ofMilliQ water, and 2.5 column volumes of 20% EtOH.

5. Column 3—Phenyl Sepharose HP Chromatography

The column dimension was as follows: INdEX100/500, 100 mmI.D.×9.7 cm=761ml. The GHPhe Buffer A consisted of 20 mM NaPO₄, 2 M NaCl, pH 7.0 with aconductivity of 163 mS/cm, and the GHPhe Buffer B consisted of 20 mMNaPO₄, pH 7.0 with a conductivity of 3.2 mS/cm. The flow rate was 90ml/min for processing, and 40 ml/min for cleaning.

The AKTA system was depyrogenated. To depyrogenate and equilibrate thePhe column, the “PheHP depy equi” program was run: the column was washedwith 2 column volumes of MilliQ water, 2 column volumes of 1 M NaOH/1 MNaCl, incubated for 30 minutes, then equilibrated with 4 column volumesof GHPhe Buffer A.

Solid NaCl was added to the GHCHTpool to 2 M. The mixture was stirred atroom temperature for 1-2 hours to dissolve, and the solution was warmedto approximately 20° C. To calculate the amount of NaCl needed (Z g):(V+Z/4000)×2×58.44=Z, or Z=116.88V/(1-116.88/4000), where V is thevolume of GHCHTpool in liters.

The GHCHTpool+NaCl mixture was loaded onto the column. The column waswashed with 3 column volumes of GHPhe Buffer A. Elution was performedwith the following complex gradient: 10% step of GHPhe Buffer B over 3column volumes, 10-80% GHPhe Buffer B gradient over 7 column volumes,80% GHPhe Buffer B step over 2 column volumes, and 100% GHPhe Buffer Bstep over 3 column volumes. The main peak was collected. The collectionwas initiated at approximately 17.3 mAU, 111 mS/cm, 46.7% GHPhe Buffer Band was ended at approx. 43 mAU, 54 mS/cm, 80% GHPhe Buffer B. Thecollected eluate was designated as GHPhe pool with the lot number, andit was a colorless solution. The next step was either performed within 2hours, or the pool was stored at 4° C. overnight. The average step yieldfrom 3 batches is 94.6%.

The Phe column was washed upflow with 2 column volumes of 1 M NaOH,incubated for 30 min, washed with 3 column volumes of GHPhe Buffer A, 3column volumes of MilliQ water, and 2.5 column volumes of 20% EtOH.After 3-5 cycles, the Phe column was washed upflow with 2 column volumesof 1 M NaOH, incubated for 30 min, washed with 3 column volumes of GHPheBuffer A, 3 column volumes of MilliQ water, 0-70% EtOH over 1 columnvolume, 3 column volumes of 70% EtOH, and stored in 20% EtOH.

6. UF/DF (Ultrafiltration/Diafiltration) II

The following filter was used for this procedure: Sartorius SartoconSlice 10 K Hydrosart cassette, 1000 cm². The GHPhe pool was concentrateddown to ˜450-ml (or ˜200 ml in the retentate flask). It was thendiafiltrated with 2.7 L (6-volumes) of GH Formulation Buffer whichconsisted of 20 mM Sodium Citrate, 20 g/L Glycine, 5 g/L Mannitol, pH6.0. The sample was concentrated down to ˜360 ml. The retentate wascollected. The system was rinsed with 300 ml of the GH FormulationBuffer, and the rinse solution was combined with the retentate. Theretentate was centrifuged at 4,000 rpm (2,862×g) for 5 minutes, and thesupernatant was collected. The supernatant was designated as Y35pAF-cBx,and was also referred to as “in-process bulk”. The in-process bulk wasaliquoted and stored at −80° C.

The overall yield of Y35pAF was 435 mg per liter of fermentation broth.The purity was >90% based on 3 HPLC methods (RP-HPLC, SEC-HPLC,IEX-HPLC) and SDS-PAGE analysis.

7. UF/DF (Ultrafiltration/Diafiltration) IIa

The following concentrator/filter was used for this procedure: AmiconStirred Cell (200 ml) with a YM10 membrane (63.5 mm). Reaction Bufferconsisted of 20 mM Sodium Acetate, 20 g/L Glycine, 5 g/L Mannitol, 1 mMEDTA, pH 4.0. A portion of in-process bulk from step 6 was used, such as250 mg of Y35pAF, and the pH was adjusted to approximately 4 by adding10-12% (v/v) of 10% acetic acid. The sample was concentrated down to25-50 ml, and Reaction Buffer was added to approximately 180 ml. Theprocess was repeated until a total of >500-fold of buffer exchange wasachieved. The sample was concentrated to approximately 25 ml. Theretentate was collected, and centrifuged at 2,000×g for 3 minutes toremove any precipitate. The supernatant was designated as Y35pAF-cBx/pH4with the date.

The protein concentration of Y35pAF-cBx/pH4 was determined by measuringA₂₇₆ of a 20-fold diluted sample, using A₂₇₆ ^(1 mg/ml)=0.818. Theconcentration of Y35pAF-cBx/pH4 was adjusted to 8 mg/ml by diluting withthe Reaction Buffer.

8. PEGylation Reaction

The amount of 30 K MPEG-Oxyamine required was calculated using the molarratio of PEG:Y35pAF=10. The PEG powder was weighed and added to the 8mg/ml Y35pAF solution at room temperature slowly, and mixed with aspatula after each addition. The reaction mixture was placed at 28° C.with gentle shaking for 18-48 hours. PEGylation was confirmed by runninga SDS gel. The reaction formed an oxime bond between hGH and PEG.

9. Column 4—Source Q Chromatography (30 m)

The column dimension was as follows: XK26/20, 26 nmI.D.×17 cm=90 ml.SourceQ Buffer A consisted of 10 mM TRIS, pH 7.0 with a conductivity of0.9 mS/cm. SourceQ Buffer B consisted of 10 mM TRIS, 1 M NaCl, pH 7.0with a conductivity of 93 mS/cm. The flow rate was 6 ml/min.

The AKTA system was depyrogenated. To depyrogenate and equilibrate theSourceQ column, the “SourceQ depy equi” was run: washed the SourceQcolumn with 2 column volumes of MilliQ water, 2 column volumes of 1 MNaOH/1M NaCl, incubated for 30 min, washed with 5 column volumes ofSourceQ Buffer B, then equilibrated with 5 column volumes of SourceQBuffer A.

20% (v/v) of 0.5 M TRIS base was added to the reaction mixture from Step8. A twenty-fold dilution was performed with 9-volumes of SourceQ BufferA and 10-volumes of MilliQ water. The mixture was then loaded onto thecolumn. The column was washed with 5 column volumes of SourceQ Buffer A.Elution was performed with a linear gradient of 0-10% SourceQ Buffer Bover 20 column volumes. The 1st major peak was collected. The collectedeluate was designated as SourceQ pool with the lot number. The pool wasstored at 4° C. overnight.

10. UF/DF (Ultrafiltration/Diafiltration) III

The following concentrator/filter was used for this procedure: AmiconStirred Cell (200 ml) with a YM10 membrane (63.5 mm). WHO Bufferconsisted of 2.5 g/L NaHCO₃, 20 g/L Glycine, 2 g/L Mannitol, 2 g/LLactose, pH 7.3.

The SourceQ pool was concentrated to 20-30 ml, and the WHO Buffer wasadded to approximately 180 ml. The process was repeated until a totalof >600-fold of buffer exchange had been achieved. The sample was thenconcentrated to 2 mg/ml or the desired concentration. The retentate wascollected, and filter sterilized with a 0.2 μm membrane in a hood. Thesterile sample was designated as PEG30-cY35pAF with the lot number.

The equivalent hGH concentration of PEG30-cY35pAF was determined bymeasuring the A₂₇₆ of diluted sample by using A₂₇₆ ^(1 mg/ml)=0.818 withtriplicate dilutions and measurements. The overall yield from Step 7 isapproximately 20%. The PEG-Y35pAF purity was >95% based on HPLC andSDS-PAGE analysis.

Example 3 hGH Purification, PEGylation, and hGH-PEG Purification ProcessPeriplasmic Preparation from E. coli

1. Periplasmic Release of hGH

An 800 gram bacterial cell pellet obtained from approximately 4 litersof fermentation broth was resuspended in 3200 ml (4-volumes) of 4-6° C.PR Buffer (50 mM TRIS, 2 mM EDTA, 0.07% Triton X-100, pH 8.0;conductivity=3 mS/cm) to obtain 20% solid. After stirring the suspensionat 4-6° C. for 1 hour, 150 ml of 8M urea was added to obtain a finalurea concentration of 0.3 M. This suspension was then stirred at 4-6° C.for 1 hour. The suspension was centrifuged at 15,000×g for 25 minutes ina J20 rotor (Avanti J20 XP centrifuge—Beckman Coulter) at 4° C. Thesupernatant was collected, and its volume measured (approximately 3.4L). The sample was designated as PRS with the date and lot number.

2. UF/DF (Ultrafiltration/Diafiltration) I

The following filter was used for this procedure: Sartorius SartoconSlice 10 K Hydrosart cassette, 1000 cm². Additional parameters include:filtrate flow rate of 80 ml/minute and TMP of approximately 14 psi.

The system was depyrogenated with 1N NaOH, and circulation allowed for30-45 minutes. The system was rinsed with approximately 2 liters ofMilliQ water until the pH dropped to below 8. Equilibration wascompleted with QFF Buffer A (10 mM Bis-TRIS, pH 6.5) for at least 5minutes. PRS was concentrated down to approximately 1.6 liters (orapproximately 1.4 liters in the retentate container). It was thendiafiltrated with 5-volumes (7 liters) of QFF Buffer A. After collectingthe retentate, the system was rinsed with 300 ml of the buffer, and therinse solution was combined with the retentate. The combined sample wasdesignated as QFFload with the lot number. It was a brownish color. Thissample was either processed within 2 hours or stored at 4° C. overnight.

The system was rinsed with MilliQ water and cleaned with 1 N NaOH bycirculating for 30-45 minutes. Rinsing was then completed with MilliQwater until the pH was less than 8. The cassette was stored in 0.1NNaOH.

3. Column 1—Q Sepharose FF Chromatography

The column dimension was as follows: 50 mm I.D.×6.3 cm=123 ml (XK26/20column). The flow rate was 35 ml/min. QFF Buffer A consisted of 10 mMBis-TRIS, pH 6.5 with a conductivity of 0.6 mS/cm. The High Salt Bufferconsisted of 10 mM TRIS, 2 M NaCl, pH 7.0 with a conductivity of 156mS/cm. QFF Buffer B consisted of 10 mM Bis-TRIS, 0.1 M NaCl, pH 6.5 witha conductivity of 11.5 mS/cm.

The AKTA system was depyrogenated. To accomplish this, the “AKTA depy”program was run three times: all buffer lines were placed in MilliQwater for the first run of the program, and then in 1 N NaOH for thesecond run. An incubation was completed for 30 minutes, and the bufferlines were placed in MilliQ water again for the third run. The program“QFF depy equi” was run to depyrogenate and equilibrate the QFF column:the QFF column was washed with 2 column volumes of MilliQ H₂O, 2 columnvolumes of 1 N NaOH/1M NaCl, incubated for 30 min, washed with threecolumn volumes of High Salt Buffer, then equilibrated with 4 columnvolumes of QFF Buffer A.

The QFFload was then loaded onto the column. The column was washed with5 column volumes of QFF Buffer A, and 5.5 column volumes of 15% QFFBuffer B in A. Elution was performed with 4.5 column volumes of 60% QFFBuffer B in A, and the elution peak was collected. The collected eluatewas designated as QFFpool with the lot number, and it was a light yellowcolor. The pool was stored at 4° C. overnight.

The column was washed with 3 column volumes of High Salt Buffer. Then 3column volumes of 1 N NaOH/1M NaCl was pumped in, and an incubation donefor 1-6 days. If the column was not used within 6 days, it was rinsedwith 1 column volume of 1 N NaOH/1M NaCl, 3 column volumes of High SaltBuffer, 3 column volumes of MilliQ H₂O, and 2.5 column volumes of 20%EtOH or 10 mM NaOH. An extensive cleaning of the column was done every3-5 cycles such that following the 1 N NaOH/1 M NaCl incubation, it waswashed upflow with 3 column volumes of Q Column Cleaning Buffer (0.5%Triton X-100, 0.1 M acetic acid), incubated for 60-80 hours, washed with1.5 column volumes of MilliQ H₂O, 1 column volume from 0 to 70% EtOH, 5column volumes of 70% EtOH, and 2.5 column volumes of 20% EtOH.

4. Column 2—Phenyl Sepharose HP Chromatography

The column dimension was as follows: 50 mm I.D.×7.5 cm=147 ml (XK26/20column). The flow rate was 35 ml/minute. Phe Buffer A consisted of 10 mMTRIS, 2 M NaCl, pH 7.0 with a conductivity of 156 mS/cm. Phe Buffer Bconsisted of 10 mM TRIS, pH 7.0 with a conductivity of 0.9 mS/cm.

The AKTA system was depyrogenated. The “AKTA depy” program was run 3times: all buffer lines were placed in MilliQ water for the 1^(st) runand then in 1 N NaOH for the 2^(nd) run. An incubation was completed for30 minutes, and then all buffer lines were placed in MilliQ water againfor the 3^(rd) run. The “PheHP depy equi” program was run todepyrogenate and equilibrate the Phe column: it was washed with 2 columnvolumes of MilliQ H₂O, 2 column volumes of 1 M NaOH/1 M NaCl, incubatedfor 30 min, then equilibrated with 4 column volumes of Phe Buffer A.

Solid NaCl was added to the QFFpool to 2 M. The mixture was stirred atroom temperature for 1-2 hours to dissolve the NaCl, and the solutionwas warmed to approximately 20° C. To calculate the amount of NaClneeded (Z g): (V+Z/4000)×2×58.44=Z, or Z=116.88V/(1-116.88/4000), whereV is the volume of QFFpool in liters.

The QFFpool+NaCl was loaded onto the column. The column was washed with5 column volumes of Phe Buffer A. Elution was performed with thefollowing complex gradient: 0-45% B linear gradient over 10 columnvolumes, 45% B step over 2 column volumes, and 100% B step over 3 columnvolumes. The main peak was collected during the gradient elution. Thecollected eluate was designated as Phe pool with the lot number, and itwas a colorless solution. The next step was performed, or the pool wasstored at 4° C.

The Phe column was washed upflow with 2 column volumes of 1 M NaOH,incubated for 30 min, washed with 3 column volumes of Phe Buffer A, 3column volumes of H₂O, and 2.5 column volumes of 20% EtOH or 10 mM NaOH.After 3-5 cycles, the Phe column was washed upflow with 2 column volumesof 1 M NaOH, incubated for 30 min, washed with 3 column volumes of GHPhe Buffer A, 3 column volumes of H₂O, 0-70% EtOH over 1 column volume,3 column volumes of 70% EtOH, and finally, stored in 20% EtOH.

5. UF/DF (Ultrafiltration/Diafiltration) II

The following filter was used for this procedure: Sartorius SartoconSlice 10 K Hydrosart cassette, 200 cm². Additional parameters include:filtrate flow rate of 15 ml/min and TMP of 14 psi. The preliminaryformulation buffer consisted of 20 mM Sodium Citrate, 20 g/L Glycine, 5g/L Mannitol, pH 6.0 with a conductivity of 4.7 mS/cm.

The system was depyrogenated with 1N NaOH, and circulation allowed for30-45 minutes. The system was rinsed with approximately 2 liters ofMilliQ water until the pH dropped to below 8. Equilibration wascompleted with Preliminary Formulation Buffer for at least 5 minutes.

The GH Phe pool was concentrated down to approximately 350 ml (orapproximately 200 ml in the retentate flask). Diafiltration wascompleted with 2.1 liters (6-volumes) of the Preliminary FormulationBuffer. Then the sample was concentrated down to approximately 350 ml,and the retentate collected. The system was rinsed with 300 ml of thebuffer, and the rinse solution was combined with the retentate. Theretentate was centrifuged at 4,000 rpm (2,862×g) for 5 minutes, and thesupernatant was collected. The supernatant was designated as Y35pAF-pBx,and was also referred to as “in-process bulk”.

The protein concentration of Y35pAF-pBx was determined by measuring A₂₇₆of diluted sample, using A₂₇₆ ^(1 mg/ml)=0.818. The in-process bulk canbe stored at 4° C. For long term storage, it was aliquoted and kept at−80° C.

The system was rinsed with MilliQ water and cleaned with 1 N NaOH bycirculating for 30-45 minutes. Then it was rinsed with MilliQ wateruntil the pH was below 8. The cassette was stored in 0.1 N NaOH.

6. UF/DF (Ultrafiltration/Diafiltration) IIa

The following concentrator/filter was used: Amicon Stirred Cell (350 ml)with a YM10 membrane (76 mm). Reaction Buffer consisted of: 20 mM SodiumAcetate, 20 g/L Glycine, 5 g/L Mannitol, 1 mM EDTA, pH 4.0 with aconductivity of 2.6 mS/cm.

The system was depyrogenated with Pyroclean. All components wereincubated in Pyroclean for 30 minutes. Rinsing with MilliQ water wascompleted until A₂₀₅ was less then 0.01.

The pH of a portion of the in-process bulk, such as 300 mg, is adjustedto approximately 4 by adding 10-12% (v/v) of 10% acetic acid. Thissample was concentrated down to 25-50 ml, and Reaction Buffer was addedto approximately 350 ml. The process was repeated until a totalof >500-fold of buffer exchange was achieved. The sample was thenconcentrated to approximately 30 ml. The retentate was collected andcentrifuged at 2,000×g for 3 minutes to remove any precipitate. Thesupernatant was designated as Y35pAF-pBx/pH4 with the date. For longterm storage, it was aliquoted and kept at −80° C.

The protein concentration of Y35pAF-pBx/pH4 was determined by measuringA₂₇₆ of a 20-fold diluted sample by using A₂₇₆ ^(1 mg/ml)=0.818. Theconcentration of Y35pAF-pBx/pH4 was adjusted to 8 mg/ml by dilution withthe Reaction Buffer.

7. PEGylation Reaction

The amount of 30 K MPEG-Oxyamine required was calculated using the molarratio of PEG:Y35pAF=5. The PEG powder was weighed and added to the 8mg/ml Y35pAF-pBx/pH4 solution at room temperature slowly while stirring.The reaction mixture was placed at 28° C. with gentle stirring for 39-50hours. The PEGylation was confirmed by performing SDS-PAGE. The reactionformed an oxime bond between hGH and PEG.

8. Column 3—Source Q Chromatography (30 cm)

The column dimension was as follows: XK26/20, 26 mm I.D.×17 cm=90 ml.The flow rate was 8 ml/minute. SourceQ Buffer A consisted of 10 mM TRIS,pH 7.0 with a conductivity of 0.9 mS/cm. SourceQ Buffer B consisted of10 mM TRIS, 1 M NaCl, pH 7.0 with a conductivity of 87 mS/cm.

To depyrogenate the AKTA system, the program “AKTA depy” was run 3times: all buffer lines were placed in MilliQ water for the 1^(st) runand in 1 N NaOH for the 2^(nd) run. An incubation was completed for 30minutes, and all buffer lines were placed in MilliQ water again for the3^(rd) run. To depyrogenate and equilibrate the SourceQ column, theprogram “SourceQ depy equi” was run: the SourceQ column was washed with2 column volumes of MilliQ H₂O, 2 column volumes of 1 M NaOH/1M NaCl,incubated for 30 minutes, washed with 5 column volumes of SourceQ BufferB, then equilibrated with 5 column volumes of SourceQ Buffer A.

20% (v/v) of 0.5 M TRIS base was added to the reaction mixture from theprevious step. A 20-fold dilution was performed with 9-volumes ofSourceQ Buffer A and 10-volumes of MilliQ H₂O. The diluted material waspassed through a 0.45 μm filter. The filtrate was then loaded onto thecolumn. The column was washed with 5 column volumes of SourceQ Buffer A.Elution was performed with a linear gradient of 0-10% SourceQ Buffer Bover 20 column volumes. The Frac-950 was used to collect elutionfractions at 13 ml/fraction. SDS-PAGE was run on the 1^(st) major peakto determine the pool. The pooled fractions were designated as SourceQpool with the lot number. The pool was stored at 4° C. overnight.

9. UF/DF (Ultrafiltration/Diafiltration) III

The following concentrator/filter was used: Amicon Stirred Cell (350 ml)with an YM10 membrane (76 mm). Preliminary Formulation Buffer consistedof 20 mM Sodium Citrate, 20 g/L Glycine, 5 g/L Mannitol, pH 6.0 with aconductivity of 4.7 mS/cm.

The system was depyrogenated with Pyroclean. All components wereincubated in Pyroclean for 30 minutes. Rinsing with MilliQ water wascompleted until A₂₀₅<0.01.

The SourceQ pool was concentrated to 20-40 ml, and the PreliminaryFormulation Buffer was added to approximately 350 ml. The process wasrepeated until a total of >600-fold of buffer exchange was achieved. Thesample was concentrated to 2 mg/ml or the desired concentration. Theretentate was collected, and filter sterilized with a 0.2 μm membrane ina hood. The sterile sample was designated as PEG30-pY35pAF with the lotnumber.

The equivalent hGH concentration of PEG30-pY35pAF was determined bymeasuring A276 of diluted sample by using A₂₇₆ ^(1 mg/ml)=0.818, andtriplicate dilutions and measurements were done. The PEG30-pY35pAF canbe stored at 4° C. For long term storage, it was aliquoted and kept at−80° C.

Periplasmic release preparations have been completed with strains ofDH10B(fis) and W3110 with the araB gene knocked out. Both strains weretransformed with orthogonal tRNA, orthogonal aminoacyl tRNA synthetase,and hGH constructs. The PEG-Y35pAF purity was >95% based on HPLC andSDS-PAGE analysis.

Example 4 Comparison of hGH Preparations: Periplasmic Release vs.Cytoplasmic (Homogenization)

FIG. 3, Panels A and B show SDS-PAGE analysis of hGH produced in E.coli. A periplasmic release batch (fermentation lot 050425B2; 800 gramsof cell paste) and a cytoplasmic batch (lysed by microfluidizer;fermentation lot 050414B1; 60 grams of cell paste) were made. Each batchwas run over a 123 ml Q FF column with QFF Buffer A consisting of 10 mMBis-TRIS, pH 6.5 and QFF Buffer B consisting of 10 mM Bis-TRIS, pH 6.5,0.1M NaCl. Three cuts were performed during elution: 15, 60, and 100%Buffer B (15 mM NaCl, 60 mM NaCl, and 100 mM NaCl respectively).Aliquots from the separation were analyzed by SDS-PAGE. The lanes forPanel A and B are as follows: lane 1=WHO hGH standard; lane 2=Load; lane3=BE/FT; lane 4=15% Buffer B; lane 5=60% Buffer B; and lane 6=100%Buffer B.

Example 5 5 Liter Fermentation Process

This example describes expression methods used for hGH polypeptidescomprising a non-natural amino acid. The strain of host cells used was amodified W3110 cell line. The host cells were transformed withconstructs for orthogonal tRNA, orthogonal aminoacyl tRNA synthetase,and a polynucleotide encoding hGH polypeptide comprising a selectorcodon. The process flow is shown as FIG. 4.

Preparation

The following reagents were prepared:

Trace Elements (Steam sterilized) Component g/l Na₃citrate 74 FeCl₃•6H₂O27 CoCl₂•6H₂O 2 Na₂MoO₄•2H₂O 2 ZnSO₄•7H₂O 3 MnSO₄•nH₂O 2 CuCl₂•2H₂O 1.3CaCl₂•2H₂O 1 H₃BO₃ 0.5

Vitamins (filter sterilized) Component g/l Niacin 6.1 Pantothenic acid5.4 Pyridoxine•HCl 1.4 Thiamine•HCl 1 Riboflavin 0.42 Biotin 0.06 Folicacid 0.04

1 M MgSO₄ (Steam sterilized) Component g/l MgSO₄•7H₂O 246

Ammonium hydroxide, 15% as NH₃ for all Component l/l 15% ammoniumhydroxide 1

Base salts, 1X, for all (steam sterilized) Component g/l Na₂HPO₄•7H₂O15.4 KH₂PO₄ 6.8 NH₄Cl 4

Concentrated feed (aseptically mixed sterile components) Component g orl per l Concentrated glycerol, 100% (w/v)  0.4 l 1 M Magnesium sulfatesolution* 0.05 l Vitamins 0.05 l Trace Elements 0.05 l Water 0.45 l*Added after steam sterilization.

Batch medium Component l/l Base salts solution, 1X 0.98 Concentratedfeed 0.02 Kanamycin stock 50 mg/ml 0.001 *: Added after steamsterilization.

Marcor Yeast extract/Glycerol mixture (Steam sterilized) Component g/lor l/l Yeast extract powder 200 g Concentrated glycerol 100% (w/v) 0.17l

Kanamycin stock for all (Filter sterilized) Component mg/ml ml Kanamycin50 3

On the day of use, the following reagents were prepared:

p-Acetyl Phenylalanine (pAF)* for all (Filter sterilized) Component g orml p-Acetyl Phenylalanine 4 g 1 M HCl 6.25 ml Water 12.5 ml *The finalvolume became 21.25 ml. Used all of the resulting solution afterfiltration.

L-(+)-Arabinose 20% for all (Filter sterilized) Component g/l mlL-(+)-Arabinose 200 1.25 *Used all of the resulting solution afterfiltration.

For each fermentation, the following was performed. 25% Struktol J 673(0.1 L) was prepared and sterilized by steam. 15% NH₃*H₂O (0.3 L) wasprepared for pH control and as nitrogen source. 10% H₃PO₄ (0.2 L) wasprepared for pH control. Concentrated feed, 1 L, was prepared in feedingcontainer 1. YE/glycerol mixture, 2 L each, was prepared in feedingcontainer 2.

The fermenter was set-up. It was sterilized with 2.5 L Base Saltssolution. The fermentor was brought to the following conditions:temperature=37° C., pH=6.9, 1.0 VVM air based on 5 L working volume (Airflow can be increased up to 2 VVM).

The concentrated feed was added to the fermentor, and 2.5 ml of 50 mg/mLkanamycin was added.

Process Schedule

Day 1 (Stage I):

About 1 ul from the E. coli MCB (master cell bank, glycerol stock) wasstabbed, and 1 μl of the glycerol stock was transferred into 2 ml batchmedium+kanamycin in a culture tube. The composition of the batch mediumwas described above.

Day 2 (Stages II and III):

Stage II:

The culture tube contained a cell density of about 1-6 (OD₆₀₀). 0.01-2ml of culture tube culture was transferred into 60 ml batchmedium+kanamycin in a 250 ml shake flask. Only healthy cells that werenot subjected to any carbon starvation were used. The composition of thebatch medium was described above.

Stage III:

The fermentor was inoculated to an initial OD₆₀₀ of about 0.05. Theamount of the cells (in L) needed from Stage 11 is

$\frac{2.5L \times 0.05}{4} = {0.031\; L\mspace{14mu}\left( {{{if}\mspace{14mu}{flask}\mspace{14mu}{OD}\; 600} = 4} \right)}$

The cells were allowed to grow batch-wise for approximately 10 hours.The specific time was dictated by the depletion of glycerol by theculture. Glycerol depletion is indicated by a sudden decrease in STIRRspeed followed by increase in pO₂ signal. The concentrated glycerol feedwas started. The feed rate was based on Formula I:

${F(0)} = {\frac{\mu_{set}}{Y_{x/s}} \times {X(0)} \times {V(0)} \times {{Exp}\left( {\mu_{set} \times 0} \right)} \times \frac{1}{S_{f}}}$

-   -   μ_(set)=0.15 h⁻¹;    -   X(0)=Yx/s*8.0 g/l    -   V(0)=2.51;    -   Exp(μ_(set)*0)=1;    -   S_(f)=400 g/l    -   The above values were inserted into the equation, F(0)=7.5 ml/h.        A scaling factor was used, so the real F(0)′ will be 8.6 ml/h.    -   F(t)′=F(0)′*Exp(μ_(set)*t). For t=13 hours, F(t)′=60.4 ml/hour.        -   The flow rate was held at 60.4 ml/hour for 1 hour.

For the run, pAF was added at the start of feed 2. Arabinose inductionwas added 1 hour after pAF addition. The final feed 2 flow rate wasmaintained until harvest.

Day 3 (Stages 1V and V)

Stage 1V.

The culture OD₆₀₀ reached about 50 to 60. At 1 hour before induction(feed time=14 hours), 1) the concentrated feed (rate=60.4 ml/hour atthis time) was stopped. 2) YE/glycerol (200 g YE and 170 g glycerol perliter) feed was started at 108.7 ml/hour. 3) A 21.25 ml bolus thatcontained 4.0 g pAF was added. Control of the pH was continued at pH6.9, using 15% ammonium hydroxide and 10% phosphoric acid to adjust pHwhen needed. 3a) The feed 2 rate was linearly increased for 3 hours,reaching 141.3 ml/hour at feed time=17 hours. The culture OUR shouldremain at about 250 mmol/l/hour. 3b) The carbon source transition wascontinued for 1 hour. 3c) An adequate amount of cells (OD₆₀₀*ml=2) wassaved for SDS-PAGE analysis.

At the time of induction (feed time=15 hours), induction was done with1.25 ml 20% (w/v or 200 g/l) L-(+)-arabinose. Adequate amounts of cells(OD₆₀₀*ml=2) were saved at 4 hours, 6 hours, and 8 hours after inductionfor SDS-PAGE analysis. The induction lasted 8 hours.

Stage V.

The culture OD₆₀₀ was checked at the end of induction. An adequateamount of cells (OD₆₀₀*ml=2) was saved for SDS-PAGE analysis. 2×200 mlculture were collected by centrifugation for evaluation by ELISA. Thecells were harvested using bucket centrifugation at 15,000 g for 22minutes, and the cells were frozen at −80° C.

This procedure has been scaled to a 100 liter culture.

Example 6 hGH Purification, PEGylation, and hGH-PEG Purification ProcessPeriplasmic Preparation from E. coli

1. Periplasmic Release of hGH

A 1.9 kilogram bacterial cell paste was resuspended in approximately 7.6liters (4-volumes) of 4° C. PR Buffer (50 mM TRIS, 10 mM EDTA, 0.07%Triton X-100, pH 8.0) to obtain 20% solid. After stirring the suspensionat 4° C. for 1 hour, 8M urea was added to obtain a final ureaconcentration of 0.3 M. The 8M urea solution was used within 48 hours ofpreparation. This suspension was then stirred at 4° C. for 1 hour. Thesuspension was centrifuged at 15,000×g for 45 minutes in a fixed angleJ20 rotor (Avanti J20 XP centrifuge—Beckman Coulter) at 4° C. Thesupernatant was collected, and its volume measured (approximately 7.7L). The sample was designated as PRS with the date and lot number.

The PRS was filtered through a prefilter, Sartopure GF2 1.2 μm capsule(1000 cm²) (Part #5571303P800B). The filtrate flow rate was 0.86 L/minat pump setting I (MasterFlex I/P Model 7529-10). The filtrate wascollected and designated as PRSF with the date and lot number. Thevolume of PRSF was measured.

The PRSF was filtered through a Sartopore 2 0.8+0.45 μm filter capsule(500 cm²) (Part #5441306G700B). The filtrate was collected anddesignated as PRSFF with the date and lot #. The volume of PRSFF wasmeasured.

In-process analysis includes non-reduced SDS-PAGE analysis to confirmthe presence of hGH in the correct form as compared with a referencestandard, measurement of A₂₇₆, and ELISA for quantitation.

2. UF/DF (Ultrafiltration/Diafiltration) I—Buffer Exchange for QFF

The following filter was used for this procedure: Sartorius SartoconSlice 10K Hydrosart cassette, 2×1000 cm². Additional parameters include:filtrate (permeate) flow rate of 100-160 ml/minute, feed pressure of24-26 psi, and retentate pressure of 5-6 psi.

The system was depyrogenated with 1N NaOH, and circulation allowed for30-45 minutes. The system was rinsed with approximately 4 liters ofMilliQ water until the pH dropped to below 8. Equilibration wascompleted with QFF Buffer A (10 mM Bis-TRIS, pH 6.5) for at least 5minutes. PRSFF was concentrated down to approximately one tenth of itsvolume. It was then diafiltrated with 8-volumes of QFF Buffer A. Theretentate was recirculated for 3-5 minutes. After collecting theretentate, the system was flushed with 300-350 ml of QFF Buffer A, andthe rinse solution was combined with the retentate. The combined samplewas filtered through a Sartopore 2 0.8+0.45 μm capsule (500 cm²) (Part#5441306G700B), and the filtrate collected was designated as QFF Loadwith the date and lot number. It was a brownish color. The volume of QFFLoad was measured, and QFF Load was either processed within 2 hours orstored at 4° C. overnight.

The system was rinsed with MilliQ water and cleaned with 1 N NaOH bycirculating for 30-45 minutes. Rinsing was then completed with MilliQwater until the pH was less than 8. The cassette was stored in 0.1NNaOH.

In-process analysis includes measurement of A₂₇₆ to quantify totalprotein and determine the amount of QFF Load for the next step, ELISA,LAL, and non-reduced SDS-PAGE analysis.

3. Column 1—Q Sepharose FF Chromatography

Q Sepharose Fast Flow was obtained from GE Healthcare. The columndimension was as follows: 70 mm I.D.×16 cm=616 ml (INdEX70/500 column).The operating capacity was 150 mg total protein (140-160 mg, based onA₂₇₆) or 10 mg GH (based on ELISA) per ml QFF. The flow rate was 100ml/min (linear velocity: 156 cm/h). QFF Buffer A consisted of 10 mMBis-TRIS, pH 6.5 with a conductivity of 0.6 mS/cm. QFF Buffer Bconsisted of 10 mM Bis-TRIS, 0.1 M NaCl, pH 6.5 with a conductivity of11.5 mS/cm.

The AKTA explorer system was depyrogenated. To accomplish this, the“AKTA depy” program was run three times: all buffer lines were placed inMilliQ water for the first run of the program, and then in 1 N NaOH forthe second run. An incubation was completed for 30 minutes, and thebuffer lines were placed in MilliQ water again for the third run. Theprogram “QFF depy equi” was run to depyrogenate and equilibrate the QFFcolumn at 30 cm/h linear velocity: the QFF column was washed with 2column volumes of MilliQ H₂O, 2 column volumes of 1 N NaOH/1M NaCl,incubated for 30 min, washed with three column volumes of Q Buffer C (10mM TRIS, 2 M NaCl, pH 7.0 with a conductivity of 156 mS/cm), thenequilibrated with 4 column volumes of QFF Buffer A.

The QFF Load was then loaded onto the column. The column was washed with4 column volumes of QFF Buffer A, and 7 column volumes of 10% QFF BufferB in A. Elution was performed with 6 column volumes of 60% QFF Buffer Bin A. The column may be washed with 3 column volumes of QFF Buffer B.The elution peak was collected. The collected eluate was designated asQFF Pool with the date and lot number. The pool was processed within 2hours or stored at 4° C. overnight.

The column was washed with 3 column volumes of Q Buffer C. Then 3 columnvolumes of 1 N NaOH/1M NaCl was pumped in, and an incubation done for1-6 days. If the column was not used within 6 days, it was rinsed with 1column volume of 1 N NaOH/1M NaCl, 3 column volumes of Q Buffer C, 3column volumes of MilliQ H₂O, and 2.5 column volumes of 20% EtOH or 10mM NaOH. An extensive cleaning of the column was done every 3-5 cyclessuch that following the 1 N NaOH/1 M NaCl incubation, it was washedupflow with 3 column volumes of Q Column Cleaning Buffer (0.5% TritonX-100, 0.1 M acetic acid), incubated for 60-80 hours, washed with 1.5column volumes of MilliQ H₂O, 1 column volume from 0 to 70% EtOH, 5column volumes of 70% EtOH, and 2.5 column volumes of 20% EtOH.

4M NaCl was added to the QFF Pool to reach a final 0.1 M concentration.The product was filtered through a Sartobind Q100X filter (Part #Q100X),pre-equilibrated with QFF Buffer B, to remove endotoxin. The filtratewas collected and labeled as QFF PoolQ with the date and lot #. Thefiltrate was processed within 2 hours or stored at 4° C. overnight.

The QFF PoolQ was passed through a Sartobran 0.45+0.2 μm filter capsule(300 cm²) (Part #5231307H500B) and the filtrate collected. The filtratewas designated QFF PoolQF with the date and lot number. The QFF PoolQFwas processed within 2 hours or stored at 4° C. overnight.

In-process analysis includes measurement of A₂₇₆, ELISA, LAL, andnon-reduced SDS-PAGE analysis.

4. Column 2—Phenyl Sepharose HP Chromatography

Phenyl Sepharose High Performance was obtained from GE Healthcare. Thecolumn dimension was as follows: 100 mm I.D.×9.7 cm=761 ml (INdEX100/500column). The operating capacity was 4.5-9 mg total protein, preferably6-8 mg total protein (based on A₂₇₆), per ml of Phenyl HP. The flow ratewas 100 ml/minute (linear velocity: 76.4 cm/h). Phe Buffer A consistedof 20 mM TRIS, 0.4 M sodium citrate pH 7.0. Phe Buffer B consisted of 10mM TRIS, pH 7.0 with a conductivity of 0.9 mS/cm.

The AKTA explorer system was depyrogenated. The “AKTA depy” program wasrun 3 times: all buffer lines were placed in MilliQ water for the 1^(st)run and then in 1 N NaOH for the 2^(nd) run. An incubation was completedfor 30 minutes, and then all buffer lines were placed in MilliQ wateragain for the 3^(rd) run. The “PheHP depy equi” program was run todepyrogenate and equilibrate the Phe column at 30 cm/h linear velocity:it was washed with 2 column volumes of MilliQ H₂O, 2 column volumes of 1M NaOH/1 M NaCl, incubated for 30 minutes, then equilibrated with 4column volumes of Phe Buffer A.

1.4 M sodium citrate was added to the QFF PoolQF to a finalconcentration of 0.4 M. The mixture was stirred at room temperature forapproximately 1 hour to dissolve the sodium citrate, and the solutionwas warmed to >16° C. The QFF PoolQF+NaCitrate was loaded onto thecolumn. The column was washed with 4 column volumes of Phe Buffer A,then 9-17 column volumes of 27% Phe Buffer B in A. The length of the 27%Phe Buffer B wash was dependent on total protein amount loaded onto thecolumn. The more protein was loaded, the less column volume of 27% PheBuffer B was required. Elution was performed with 8-10 column volumes of48% Phe Buffer B in A. The column was washed again with 100% Phe BufferB. The 48% B elution peak was collected and designated as Phe Pool withthe lot number. The next step was performed within 2 hours, or the poolwas stored at 4° C. overnight.

The Phe column was washed upflow with 2 column volumes of 1 M NaOH,incubated for 30 minutes, washed with 3 column volumes of Phe Buffer A,3 column volumes of H₂O, and 2.5 column volumes of 20% EtOH or 10 mMNaOH. After 3-5 cycles, the Phe column was washed upflow with 2 columnvolumes of 1 M NaOH, incubated for 30 minutes, washed with 3 columnvolumes of Phe Buffer A, 3 column volumes of H₂O, 0-70% EtOH over Icolumn volume, 3 column volumes of 70% EtOH, and finally, stored in 20%EtOH or 10 mM NaOH.

In-process analysis includes measurement of A₂₇₆, ELISA, LAL, andnon-reduced SDS-PAGE analysis.

5. UF/DF II—Formulation of In-Process Bulk GH

The following filter was used for this procedure: Sartorius SartoconSlice 10K Hydrosart cassette, 1000 cm². Additional parameters include:filtrate (permeate) flow rate of 50-90 ml/min, feed pressure of 20-27psi, and retentate pressure of 3-4 psi. The UF/DF II Buffer consisted of10 mM Sodium Phosphate, 20 g/L Glycine, and 5 g/L Mannitol, pH 7.0.

The system was depyrogenated with 1N NaOH, and circulation allowed for30-45 minutes. The system was rinsed with approximately 4 liters ofMilliQ water until the pH dropped to below 8. Equilibration wascompleted with UF/DF II Buffer for at least 5 minutes.

The Phe Pool was concentrated down to approximately 700-900 ml (orapproximately 500-700 ml in the retentate flask). Diafiltration wascompleted with 4.2-5.4 liters (6-volumes) of the UF/DF II Buffer. Theretentate was recirculated for 3-5 minutes, and the retentate wascollected. The system was flushed with 100-200 ml of UF/DF II Buffer,and the rinse solution was combined with the retentate. The combinedsample was filtered with a Sartobran 0.45+0.2 μm capsule (150 cm²) (Part#5231307H400B), and the filtrate was designated as Y35pAF-pBx and wasalso referred to as “in-process bulk.”

The protein concentration of Y35pAF-pBx was determined by measuring A₂₇₆of a diluted sample, using A₂₇₆₁ mg/ml=1.037. The in-process bulk can bestored at 4° C. for up to 1 week. For long term storage, it wasaliquoted and kept at −80° C.

The system was rinsed with MilliQ water and cleaned with 1 N NaOH bycirculating for 30-45 minutes. Then it was rinsed with MilliQ wateruntil the pH was below 8. The cassette was stored in 0.1 N NaOH.

In-process analysis includes RP-HPLC, measurement of A₂₇₆, ELISA, LAL,and non-reduced SDS-PAGE analysis.

6. UF/DF IIa—Concentration and Buffer Exchange for PEGylation

The following filter was used for this procedure: Sartorius SartoconSlice 10 K Hydrosart cassette, 200 cm². Additional parameters include:filtrate (permeate) flow rate of 12-14 ml/min, feed pressure ofapproximately 25 psi, and retentate pressure of 0-0.5 psi. The ReactionBuffer consisted of 20 mM Sodium Acetate, 20 g/L Glycine, 5 g/LMannitol, 1 mM EDTA, pH 4.0 with a conductivity of 2.6 mS/cm.

The system was depyrogenated with 1N NaOH and circulation allowed for30-45 minutes. The system was rinsed with approximately 2 liters ofMilliQ water until the pH dropped to below 8. Equilibration wasperformed with Reaction Buffer for at least 5 minutes.

The pH of an amount of the in-process bulk from step 5 was adjusted toapproximately 4 by adding 3.7% (v/v) of 10% acetic acid. Then it wasconcentrated down to the target volume with 8 mg/ml concentration basedon the amount of starting hGH used. The sample was then diafiltered with5 volumes of Reaction Buffer. The retentate was recirculated for 3-5minutes, and then retentate was collected. The system was flushed with80-120 ml of Reaction Buffer and combined with the retentate. Thecombined retentate was filtered through a Sartobran 0.45+0.2 μm capsule(150 cm²) (Part #5231307H400B). The filtrate was designated asY35pAF-pBx/pH4 with the date. The sample can be stored at 4° C.overnight.

The protein concentration of Y35pAF-pBx/pH4 was determined by measuringA₂₇₆ of a 20-fold diluted sample using A₂₇₆ ^(1 mg/ml)=1.037. Theconcentration of Y35pAF-pBx/pH4 was adjusted to 7 mg/ml (5-9 mg/ml) bydilution with the Reaction Buffer.

7. PEGylation Reaction

The molecular weight of hGH with the p-acetyl-phenylalanine substitutedfor the tyrosine at position 35 (Y35pAF) was 22,149 Da, and themolecular weight of lot of mPEG-oxyamine was 30,961 Da. See SEQ ID NO: 2of US Patent Publication No. 2005/0170404 for the sequence of wild-typemature hGH. FIG. 5 shows the chemical structure of the PEG used. Usingthe molar ratio of PEG:Y35pAF=5, the amount of 30 K MPEG-Oxyaminerequired was calculated. The PEG powder was weighed and added to the 7mg/ml Y35pAF solution at 25-28° C. slowly while stirring. Large piecesof solid PEG were manually broken up. Following the last addition, thereaction mixture was placed at 28° C. with gentle stirring for 39-50hours. The reaction formed an oxime bond between hGH and PEG.

In-process analysis includes non-reduced SDS-PAGE analysis to confirmthe PEGylation.

8. Column 3—Source 30Q Chromatography

Source 30Q was obtained from GE Healthcare. The column dimension was asfollows: 70 mm I.D.×17.5 cm=673 ml (INdEX 701500 column). The operatingcapacity was 2.4 mg (1-2.8 mg) GH per ml SourceQ. The flow rate was 80ml/minute (linear velocity: 125 cm/h). SourceQ Buffer A consisted of 5mM TRIS, pH 7.0. SourceQ Buffer B consisted of 5 mM TRIS, 0.1 M NaCl, pH7.0.

To depyrogenate the AKTA explorer system, the program “AKTA depy” wasrun 3 times: all buffer lines were placed in MilliQ water for the 1^(st)run and in 1 N NaOH for the 2^(nd) run. An incubation was completed for30 minutes, and all buffer lines were placed in MilliQ water again forthe 3^(rd) run. To depyrogenate and equilibrate the SourceQ column, theprogram “SourceQ depy equi” was run: the SourceQ column was washed with2 column volumes of MilliQ H₂O, 2 column volumes of 1 M NaOH/1M NaCl,incubated for 30 minutes, washed with 5 column volumes of SourceQ BufferB, then equilibrated with 5 column volumes of SourceQ Buffer A.

20% (v/v) of 0.5 M TRIS base was added to the reaction mixture from theprevious step (step 7). The sample was then passed through a Sartobran0.45+0.2 μm filter capsule (150 cm²) (Part #5231307H400B). A 20-folddilution was performed with 9-volumes of SourceQ Buffer A and 10-volumesof MilliQ H₂O. The diluted sample was then loaded onto the column. Thecolumn was washed with 5 column volumes of SourceQ Buffer A. Elution wasperformed with a linear gradient of 0-50% SourceQ Buffer B over 10column volumes. Fractions were collected at approximately ⅕ columnvolume/fraction. SE-HPLC and non-reduced SDS-PAGE analysis wereperformed on the 1^(st) major peak to determine the pool. The pooledfractions were designated as SourceQ pool with the date and lot number.The pool was stored at 4° C. overnight.

9. UF/DF (Ultrafiltration/Diafiltration) III—Concentrate and BufferExchange for Formulated Bulk

The following filter was used: Sartorius Sartocon Slice 10K Hydrosartcassette, 200 cm². Additional parameters include: filtrate (permeate)flow rate of 12-14 ml/min, feed pressure of approximately 25 psi, andretentate pressure of approximately 0-0.5 psi.

The system was depyrogenated with 1 N NaOH, and circulation was allowedfor 30-45 minutes. The system was rinsed with approximately 2 liters ofMilliQ water until the pH dropped to below 8. Equilibration was thenperformed with Formulation Buffer for at least 5 minutes.

The SourceQ pool (step 3.6) was concentrated to the target volume of 8mg/ml concentration based on the amount of starting material used.Diafiltration was performed with 6-volumes of Formulation Buffer. Theretentate was recirculated for 3-5 minutes, and the retentate wascollected. The system was flushed with 50-100 mls of Formulation Bufferand combined with the retentate. The combined retentate was sterilefiltered with a Sartobran 0.45+0.2 μm capsule (150 cm²) (Part#5231307H400B) using sterile technique in a biosafety hood or a Class100 hood. The sterile sample was designated as PEG30-pY35pAF with thelot number.

The equivalent hGH concentration of PEG30-pY35pAF was determined bymeasuring A₂₇₆ of a diluted sample by using A₂₇₆₁ mg/ml=1.145 withtriplicate dilutions and measurements. The PEG30-pY35pAF can be storedat 4° C. for up to 3 days. For long term storage, it was aliquoted andkept at −80° C.

Material from a strain of W3110 has been processed with this protocol.The strain used was transformed with orthogonal tRNA, orthogonalaminoacyl tRNA synthetase, and hGH constructs. The PEG-Y35pAF puritywas >95% based on HPLC and SDS-PAGE analysis.

Full release assays include, but are not limited to, assays thatevaluate attributes of PEG30-pY35pAF such as appearance, dissolve time,identity and purity, potency, safety, and other attributes including,but not limited to, pH. Test methods for evaluation include, but are notlimited to, reduced and non-reduced SDS-PAGE, SE-HPLC, RP-HPLC,IEX-HPLC, CEX-HPLC, measurement of host cell protein, measurement ofresidual DNA, A₂₇₆ for concentration, cell proliferation assays, LAL,pyrogen, sterility, bioburden (microbial limit), Karl Fisher (watercontent), content uniformity, and osmolality.

The buffer used in the buffer exchange of UF/DF III may be any suitablebuffer. Additional steps after UF/DF III include, but are not limitedto, lyophilization. Lyophilization can be done using standard techniquesknown to those of ordinary skill in the art.

This method has been performed with a bacterial cell pellet of about 2.7kg.

Example 7 Additional Methods

Purity Analysis by SDS-PAGE

The following method was used to evaluate the purity of in-process andfinal bulk recombinant hGH and PEG-recombinant hGH conjugates bySDS-PAGE, followed by total protein staining. Any charged molecule suchas a protein will migrate when placed in an electric field. The velocityof migration of a protein in an electric field depends on the strengthof the electric field, the net electric charge on the protein, and thefrictional resistance. The frictional resistance is the function of thesize and shape of the protein. When denatured in the presence of excessSDS, most proteins bind SDS in a constant weight ratio such that theyhave essentially identical charge densities and migrate inpolyacrylamide gels according to protein size. Proteins separated by gelelectrophoresis can be detected by Coomassie Brilliant Blue staining.

Equipment for this procedure included, the following or equivalentsthereof: XCell Surelock Mini-Cell (Invitrogen), heat block set to+70-80° C., power supply (up to 200V), microcentrifuge (such as BeckmanCoulter Microfuge 18 or 22R), and reciprocal shaker. Reagents includedNuPAGE MOPS SDS Running Buffer (20×, Invitrogen PN NP0001); NuPAGE MESSDS Running Buffer (20×, Invitrogen PN NP0002); NuPAGE LDS Sample Buffer(4×, Invitrogen PN NP0007); NuPAGE Sample Reducing Agent (10×,Invitrogen PN NP0009); 12% Bis-Tris NuPAGE precast gel, 1.0 mm×10-well(Invitrogen PN NP0341BOX); 4-12% Bis-Tris NuPAGE precast gel, 1.0mm×10-well (Invitrogen PN NP0321BOX); Pre-Stained Molecular WeightMarker (SeeBlue Plus2, Invitrogen PN LC5925); MilliQ-quality H₂O orequivalent; SimplyBlue SafeStain (Invitrogen PN LC6065) or equivalent;reference standard (WHO rhGH standard; calibration solutions forrhGH(Y35pAF-pB2/pB3, 2 mg/ml); calibration solutions for the pEG-rhGHconjugate (PEG30-pY35pAF-01, 2 mg/mL). Protein concentrations of thestandards and the test article were measured using standard techniquesknown in the art.

Analysis of pre-PEGylation purification step samples

3 μg reference standard (RS, e.g. calibration solution Y35pAF-pB2/pB3)were prepared under non-reducing conditions. 3 μg of reference standardwas added to 4×LDS and MilliQ H₂O to obtain a 28 μl sample in 1X LDS.Similarly, the rhGH test article was prepared under non-reducingconditions. Both the rhGH test article and reference standards wereheated at +70-80° C. for 8-10 minutes and centrifuged prior to loadingonto the gel. The 12% Bis-Tris NuPAGE precast gel was prepared with 1XMOPS SDS Running Buffer according to manufacturer's instructions. Thegel was loaded as follows: Pre-Stained Molecular Weight Marker, 3 μgreference standard, test articles and run with a maximum setting of 200Vfor 50 minutes. The gel was incubated in di-H₂O, stained with shakingusing SimplyBlue or an equivalent, and destained with water. The majorband position of the rhGH test article is compared to that of the 3 μgreference standard.

Analysis of the Purified in-Process Bulk rhGH

20 μg and 1 μg of the reference standard (RS, e.g WHO rhGH) wereprepared under non-reducing and reducing conditions. For non-reducedconditions, 20 or 1 μg of reference standard was added to 4×LDS andMilliQ H₂O to obtain a 28 μl sample in 1X LDS. For reduced conditions,20 or 1 μg of reference standard was added to 4×LDS, 10X Reducing Agent,and MilliQ H₂O to obtain a 28 μl sample in 1X LDS and 1X Reducing Agent.Both the rhGH test articles and reference standards were heated at+70-80° C. for 8-10 minutes and centrifuged prior to loading onto thegel. 12% Bis-Tris NuPAGE precast gels were run in 1X MOPS SDS RunningBuffer according to manufacturer's instructions with one unit for thenon-reducing condition and the other unit for the reducing condition.The gels were loaded as follows: Pre-Stained Molecular Weight Marker, 1μg reference standard, 20 μg reference standard, blank lane, followed bythe test articles at a maximum setting of 200V for 50 minutes. The gelswere incubated in di-H₂O, stained with shaking using SimplyBlue or anequivalent, and destained with water. The major band position of therhGH test article is compared to the 20 μg reference standard. In thelane of the rhGH test article, no band apart from the major band shouldbe more intense than the major band in the lane of the 1 μg referencestandard (5%).

Analysis of PEGylation of rhGH and Purification of PEG-rhGH

The reference standard (RS, e.g. calibration solution PEG30-pY35pAF-01)was prepared under non-reducing conditions. 5 μg of PEG30-pY35pAF-01 wasadded to 4×LDS and MilliQ H₂O to obtain a final 28 μl sample in 1X LDS.5-20 μg of the test article, depending on the procedure being analyzed,was added to 4×LDS and MilliQ H₂O to obtain a final 28 μl sample in1×LDS. For the PEGylation reaction mixtures, 15-20 μg of the testarticle was used. For the analysis of the PEGylation reaction mixture, acomparison was made between: a) serial concentrations of the rhGH priorto the additional of PEG at pH 4 to allow estimation of the relativepercent of non-PEGylated rhGH remaining in the PEGylation reactionmixture; b) 10 μL of a 1/10 dilution of the reaction mixture. 5-20 μg ofthe test article was used from column fractions during the purificationpost PEGylation. For the analysis of the PEG-rhGH column fractions,column fractions were compared by using fixed volumes of each columnfraction (typically 21 μL of each column fraction).

PEG-rhGH test articles or PEG-rhGH reference standard samples were notheated. Samples were centrifuged and loaded onto a 4-12% Bis-Tris NuPAGEprecast gel prepared with 1X MES SDS Running Buffer according tomanufacturer's instructions. The gel was loaded as follows: Pre-StainedMolecular Weight Marker, 5 μg reference standard, followed by the testarticles and run with a maximum setting of 200V for 35 minutes. The gelswere incubated in di-H₂O, stained with shaking using SimplyBlue or anequivalent, and destained with water.

The electropherogram of the PEG-rhGH test article should conform to theelectropherogram obtained with the PEG-rhGH reference standard.

Analysis of final PEGylated rhGH product

10 μg of the reference standard (RS, e.g. calibration solutionPEG30-pY35pAF-01) was prepared under non-reducing and reducingconditions. 10 ug of PEG30-pY35pAF-01 (2 mg/mL) was added to 4×LDS andMilliQ H₂O to obtain a final 28 μl sample in 1X LDS. For reducedconditions, 10 μg of reference standard was added to 4×LDS, 10× ReducingAgent, and MilliQ H₂O to obtain a 28 μl sample in 1×LDS and 1X ReducingAgent. Similarly, 10 g of pegylated rhGH test articles were alsoprepared under non-reduced and reduced conditions. The PEG-rhGH testarticles and PEG-rhGH reference standards were not heated, but were snapcentrifuged prior to loading on 4-12% Bis-Tris NuPAGE precast gelsprepared with 1X MES SDS Running Buffer according to manufacturer'sinstructions. The gels were loaded in the order of Pre-Stained MolecularWeight Marker, 10 μg reference standard, blank lane (recommended tominimize potential carryover effects), followed by the test articleswith a maximum setting of 200V for 35 minutes. The gels were incubatedin di-H₂O, stained with shaking using SimplyBlue or an equivalent, anddestained with water.

The electropherogram of the PEG-rhGH test article should conform to theelectropherogram obtained with the PEG-rhGH reference standard. Theelectropherogram of the PEG-rhGH test article should conform to theelectropherogram obtained with the PEG-rhGH reference standard. Anybands that do not match the reference standard may be degradationproducts or aggregates. Higher molecular weight bands may representaggregates, and lower molecular weight bands may represent polypeptidethat is no longer conjugated to PEG.

Purity and Chemical Degradation Analysis of rhGH by CEX-HPLC/IEX-HPLC

The following method was used to assess relative purity and potentialchemical degradation (i.e. deamidation) of PEGylated recombinant humangrowth hormone (rhGH) by cation-exchange high performance liquidchromatography (CEX-HPLC). CEX-HPLC is a technique that relies oncharge-charge interactions between a protein and the charges immobilizedon the resin. Cation exchange chromatography takes advantage of thepositively charged ions of a protein that bind to the negatively chargedresin. A common structural modification of rhGH deamidation ofasparagine (Asn) residues, and this CEX-HPLC method permits theseparation of deamidated and deamidation intermediates of PEGylated andnonPEGylated rhGH. This method was used to support identification andpurity assessment of PEGylated rhGH. Some partial degradation productsof rhGH are observable using this technique.

Equipment for this procedure included, the following or equivalentsthereof: UV/Vis Spectrophotometer (Agilent 8453 or equivalent); 50 μlquartz cuvette; 0.5 mL Vivaspin concentrators (if needed; Vivascience10,000 MWCO, PES, VS0102 or equivalent); PD-10, NAP-10, or NAP-5 column(GE Healthcare, Cat. #17-0851-01, 17-0853-01, 17-0854-01); HPLC vialsand caps (Alltech 100 μl screw cap polypropylene vials #12962, TFE linercaps #73048, open hole screw caps #73044, or equivalent); clean 1 and 2L glass bottles; column—PolyCAT A 4.6×200 mm, 5μ, 1000 Å (PolyLC,204CT0510) and PolyCAT A guard column, 4.6×10 mm, 5μ, 1000 Å (PolyLC,JGCCT0510); high-pressure liquid chromatography instrument capable ofperforming linear gradients (such as Agilent 1100 HPLC equipped with avacuum degasser, quaternary pump, thermostatted autosampler,thermostatted column compartment, diode array detector (DAD), andChemstation chromatography software).

Reagents for this procedure included water (Milli-Q quality orequivalent) and solid chemicals are analytical grade or better andsolvents are HPLC grade or better, unless otherwise noted. Storage ofreagents and procedural steps occurred at room temperature, unlessotherwise indicated. Examples of such chemicals include AmmoniumAcetate, Spectrum A2149, HPLC grade, or equivalent; Acetonitrile, FisherA998; HPLC grade, or equivalent; Ammonium Bicarbonate, Fluka #09830,Ultra>99.5%, or equivalent; Glacial Acetic Acid, Fisher #64-19-7, HPLCgrade, or equivalent; Sodium Citrate Dihydrate, Spectrum S0165, USPgrade, or equivalent; Glycine, Spectrum AM125 or equivalent; Mannitol,Spectrum MA165, or equivalent; 6N HCl, Mallinckrodt 2662-46, orequivalent.

Mobile phase A buffer was 50 mM Ammonium Acetate, pH 4.25, 40%Acetonitrile (AcCN), and Mobile Phase B buffer was 500 mM AmmoniumAcetate, pH 4.25, 40% AcCN. Additional reagents prepared were 10% aceticacid; Buffer for Deamidation: 30 mM Ammonium Bicarbonate, pH 9.0; andSample Dilution Buffer: 20 mM Sodium Citrate, 20 g/L Glycine, 5 g/LMannitol, pH 6.0, each sterile filtered using 0.22 μm PES filters(Corning #431098, or equivalent).

World Health Organization (WHO) rhGH (Cat. #98/574) was used as anon-PEGylated hGH standard. It was reconstituted in 1.0 ml of water anddiluted to 1.1 mg/ml using dilution buffer. 10% (v/v) of 10% acetic acidwas added to bring the pH between pH 3.8-4.3 with a final concentrationof 1.0 mg/ml (acceptable range 0.9-1.1 mg/ml). Another non-PEGylated hGHstandard, the calibration solution Y35pAF-pB2/pB3, was prepared in asimilar fashion. A PEGylated hGH standard, calibration solutionPEG30-pY35pAF-01, was also prepared in a similar fashion.

For the PEGylated Resolution Solution, the PEG30-pY35pAF-01 calibrationsolution was buffer exchanged into 30 mM Ammonium Bicarbonate, pH 9.0buffer using a PD-10, Nap-10, or Nap-5 desalting column. The standardwas concentrated using a 0.5 mL Vivaspin concentrator to approximately 2mg/ml (acceptable range 1.9-2.1 mg/ml), and the sample was incubated at37° C. for 24 hours. The sample or portion of the sample needed wasdiluted to 1.1 mg/ml using dilution buffer, and 10% (v/v) of 10% aceticacid was added to bring pH between pH 3.8-4.3 with a final concentrationof 1.0 mg/ml (acceptable range 0.9-1.1 mg/ml).

The test article was diluted to 1.1 mg/ml using dilution buffer and 10%(v/v) of 10% acetic acid was added to bring pH between pH 3.8-4.3 with afinal concentration of 1.0 mg/ml (acceptable range 0.9-1.0 mg/ml).Protein concentrations of the standards and the test article weremeasured using standard techniques known in the art.

Procedure

The instrument was set-up with the following conditions: 1) Column:PolyCAT A 204CT0510 and JGCCT0510; 2) Auto sampler Temperature: roomtemperature; 3) Pump Setup: step gradient: 81.5-108.5 mM AmmoniumAcetate pH 4.25 (7-13% B), followed by 108.5-500 mM Ammonium Acetate pH4.25 (13-100% B); 4) Table 4;

TABLE 4 Pressure Time Mobile Phase A Mobile Phase B Flow (ml/min) (bar)0 100 0 1.0 140 10 100 0 1.0 140 11 93 7 1.0 140 91 87 13 1.0 140 102 0100 1.0 140 118 0 100 1.0 140 119 100 0 1.0 140 151 100 10 1 1405) Injector Setup—Injection: Standard Injection; Injection Volume: 25μl; Draw Speed: 50 μl/min; Injection Speed: 50 μl/min; Needle wash: 15μl H₂O; Stop Time: As pump; 6) DAD signals: Table 5;

TABLE 5 Sample Bw Reference Bw Units 280 4 600 100 nm 276 4 600 100 nm214 8 600 100 nm 220 4 600 100 nm 250 8 600 100 nmPeak Width: >0.1 min; Slit: 4 nm; Stop Time: as pump; 7) ColumnThermostat: Temperature: 30° C.; record the temperature.

The column was equilibrated with 10-15 column volumes of 100% mobilephase A. 25-50 μl of the PEGylated calibration solution PEG30-pY35pAF-01was injected. The main PEGylated peak eluted at a retention time of56.97 min (+0.5 min). Next, 25-50 μl of the WHO or calibration solutionY35pAF-pB2/pB3 was injected and the HPLC program was run. The mainnon-PEGylated peak eluted at a retention time of 98.54 min (0.5 min), arelative retention time of 1.73±0.01 to the main PEGylated peak.

25-50 μl of the PEGylated resolution solution was then injected. In thechromatogram obtained, the main PEGylated peak eluted at a retentiontime of 56.97 min (+0.5 min), and the PEGylated deamidated peak elutedat a retention time of 0.79±0.02 relative to the main peak (45.23±0.3min; (current conditions result in a resolution of 2.3±0.02).

25-50 μl of the PEGylated test article was then injected, and the HPLCprogram was run. The samples were run in triplicate, and averageretention times were noted. Chromatograms were generated with absorbance(280 nm).

Data Analysis

The retention time of the PEGylated rhGH test article was compared withthe calibration solution PEG30-pY35pAF-01. The average purity of thetest article was calculated using: (Integration area of the mainpeak/integration areas of all peaks)×100%. Any peak(s) due to thesolvent were disregarded.

Purity Determination of rhGH by SEC-HPLC

This procedure was used to assess the purity of recombinant human growthhormone (rhGH) including in-process material and PEGylated rhGH bysize-exclusion high performance liquid chromatography (SEC-HPLC). Thistest separates monomer from dimer and other related substances of highermolecular weight in the sample, as well as PEGylated and nonPEGylatedsamples. SEC-HPLC is a technique using the stationary phase as a porousmatrix which is permeated by mobile phase molecules. Sample moleculessmall enough to enter the pore structure are retarded, while largermolecules are excluded and therefore rapidly carried through the column.Thus, size exclusion chromatography means separation of molecules bysize and the chromatographic elution time is characteristic for aparticular molecule. This procedure is used to determine the percentageof monomer (PEGylated and unpEGylated) rhGH. Dimer and other highmolecular weight proteins are observable using this technique.

References for this technique include European Pharmacopoeia 2002, p.193; British Pharmacopoeia 2001, p. 1941; “High-PerformanceSize-Exclusion Chromatographic Determination of the Potency ofBiosynthetic Human Growth Hormone Products” by R. M. Riggin et al.Journal of Chromatography 435 (1988), p. 307-318.

Equipment for this procedure included, the following or equivalentsthereof: UV/Vis Spectrophotometer (Agilent 8453 or equivalent); 50 ulquartz cuvette; 0.5 mL Vivaspin concentrators (if needed; Vivascience10,000 MWCO, PES, VS0102 or equivalent); HPLC vials and caps (Alltech100 ul screw cap polypropylene vials #12962, TFE liner caps #73048, openhole screw caps #73044, or equivalent); clean 1 and 2 L glass bottles;Column—Tosohaas TSK Super SW3000 18675 and Super SW Guard Column 18762,a silica-based size exclusion HPLC column with a dimension of 4.6×300mm, particle size of 4 μm and pore size of 250 A along with a guardcolumn having a dimension of 4.6×35 mm and 4μ particle size;High-pressure liquid chromatography instrument capable of performinglinear gradients (such as Agilent 1100 HPLC equipped with a vacuumdegasser, quaternary pump, thermostatted autosampler, thermostattedcolumn compartment, diode array detector (DAD), Refractive Indexdetector (RID) and Chemstation chromatography software).

Reagents for this procedure included water (Milli-Q quality orequivalent) and solid chemicals are analytical grade or better andsolvents are HPLC grade or better, unless otherwise noted. The storageof reagents and procedural steps occurred at room temperature, unlessotherwise indicated. Examples of such chemicals included MonobasicMonohydrate Sodium Phosphate, Spectrum U.S.P. grade S0130, orequivalent; Dibasic Heptahydrate Sodium Phosphate, Spectrum U.S.P. gradeS0140, or equivalent; 2-propanol, Fisher HPLC grade A451-4, orequivalent.

Mobile phase buffer was 97% of 63 mM sodium phosphate pH 7.0, 3% of2-propanol. Solution A was 25 mM Sodium Phosphate, pH 7.0. Both weresterile filtered using 0.22 μm PES filters (Corning #431098, orequivalent).

World Health Organization (WHO) rhGH (Cat. #98/574) was used as anon-PEGylated hGH standard. It was reconstituted with 1.0 ml of waterand diluted to 1 mg/ml concentration (acceptable range 0.9-1.1 mg/ml) inWHO buffer. Another non-PEGylated hGH standard, calibration solutionY35pAF-pB2/pB3, was prepared in a similar fashion and diluted with 20 mMsodium citrate, 2% glycine, 0.5% mannitol, pH 6. A PEGylated hGHstandard, calibration solution PEG30-pY35pAF-01, was also prepared in asimilar fashion and diluted with 20 mM sodium citrate, 2% glycine, 0.5%mannitol, pH 6. For the Resolution Solution, the PEG30-pY35pAF-02 highermolecular weight standard was brought to 1 mg/ml concentration(acceptable range 0.9-1.1 mg/ml). This solution contains approximately33% PEG-PEG-GH, 66.5%-GH). Test material was diluted to approximately1.0 mg/ml with Solution A (acceptable range 0.9-1.1 mg/ml). All sampleconcentrations were measured using standard techniques known in the art.The dilution of samples may be performed with any suitable buffer.

Procedure

The instrument was set-up with the following conditions: 1) Column: TSKSuper SW3000 18675 and Guard Column 18762; 2) Pump Setup—gradient:isocratic; flow rate: 0.3 ml/min; duration: 25 min; Max Pressure: 120bar; 3) Injector Setup—Injection: Standard Injection; Injection Volume:10 μl; Draw Speed: 100 μl/min; Injection Speed: 100 μl/min; Needle wash:100 ul H₂O; Stop Time: As pump; 4) DAD Signals: Table 6;

TABLE 6 Sample Bw Reference Bw Units 214 4 600 100 nm 276 4 600 100 nm220 8 600 100 nm 280 4 600 100 nm 250 8 600 100 nmPeak Width:>0.05 min; Slit: 2 nm; Stop Time: as pump; 5) RIDSignal—Temperature: 35° C.; Response Time: >0.2 min 4s, standard; 6)Column Thermostat: Temperature: 23° C.; record the temperature.

The column was equilibrated with 10 column volumes (50 ml=166 min at 0.3ml/min) of the mobile phase, and the RID was purged for at least 20minutes before injecting samples. DAD and RI detectors were autobalancedbefore sample runs.

20 μl of the calibration solution Y35pAF-pB2/pB3 (or WHO standard) wasinjected, and the HPLC program was run. In the chromatogram obtained,the main unPEGylated peak eluted at a retention time of approximately12.96 (+0.05) min. The higher molecular weight unPEGylated rhGH dimereluted at a retention time of 0.94±0.02 relative to the main peak.Higher molecular weight aggregates eluted at retention times of 7.3-8.0min.

20 μl of the calibration solution PEG30-pY35pAF-01 was injected. Themain pegylated peak eluted at a retention time of approximately 8.33(±0.08) min (relative retention time of 0.64 to the unPEGylated rhGH).Higher molecular weight PEGylated rhGH aggregates eluted at timesgreater than 8.0 min.

20 μl of the resolution solution was injected, and the HPLC program wasrun. The main PEGylated peak elutes at a retention time of 8.28 min, andthe higher molecular weight species eluted at 7.54 min, a relativeretention time of 0.9 (0.05) relative to the main PEGylated peak.

20 μl of the test article was injected, and the HPLC program was run.Samples were run in triplicate and average retention times were noted.The retention time of the rhGH test article was compared with the rhGHstandard(s).

The SEC-HPLC data from the test article was compared to data obtainedfrom the reference standards. To determine the purity of non-PEGylatedrhGH, the integrated main peak areas of the rhGH test article wascompared with the total peak area, and the percentage of monomer in therhGH test article was calculated by: (main peak area of rhGHsample/total peak area)×100%. The percentage of dimer and/or higheraggregates were calculated in the hGH test article. Any peak(s) due tothe solvent were disregarded. To determine the purity of PEGylated rhGH,the integrated main peak areas of the PEGylated rhGH sample was comparedwith the total peak area, and the percentage of PEGylated monomer inPEG-rhGH sample was calculated by: (main peak area of PEG-rhGHsample/total peak area)×100%. The percentage of PEGylated dimer, higheraggregates, and nonPEGylated monomer were calculated in the PEGylatedhGH test article. Any peak(s) due to the solvent were disregarded. Peakseluting in the chromatogram prior to the main PEGylated hGH peakrepresent higher molecular weight species. Such higher molecular weightspecies may include but are not limited to dimers (such as PEG-PEG-hGHand other possible dimers) or soluble aggregates. Peaks eluting afterthe main PEGylated hGH peak represent lower molecular weight species.Such lower molecular weight species may include but are not limited tonon-PEGylated monomer and clipped forms of PEGylated hGH.

Purity and Chemical Degradation Analysis of rhGH by RP-HPLC

The following method was used to assess relative purity and potentialchemical degradation (deamidation and oxidation) of recombinant humangrowth hormone (rhGH) by C4 reverse phase high performance liquidchromatography (RP-HPLC). RP-HPLC is a technique that separatesmolecules on the basis of relative hydrophobicities. Samples are passedover a stationary phase of silica covalently bonded to hydrocarbonchains. The molecules of interest are retarded by the stationary phaseand eluted with an isocratic solvent. The chromatographic elution timeis characteristic for a particular molecule. This method separates rhGHbased on subtle differences in hydrophobicity and retention behaviorassociated with structural modifications such as deamidation. Thismethod was used to support identification and purity assessment of rhGH.Some partial degradation products of rhGH are observable using thistechnique.

References for this technique include European Pharmacopoeia 2002, p.193; British Pharmacopoeia 2001, p. 1938-1939; A Reversed-Phase HighPerformance Liquid Chromatographic Method for Characterization ofBiosynthetic Human Growth Hormone” by R. M. Riggin et al. AnalyticalBiochemistry 167, 199-209 (1987).

Equipment for this procedure included, the following or equivalentsthereof: UV/Vis Spectrophotometer (Agilent 8453 or equivalent); 50 ulquartz cuvette; PD-10, Nap-10, or Nap 5 (depending on sample volume; GEHealthcare Nap5 column 17-0853-02 or equivalent); 0.5 mL Vivaspinconcentrators (if needed; Vivascience 10,000 MWCO, PES, VS0102 orequivalent); HPLC vials and caps (Alltech 100 ul screw cap polypropylenevials #12962, TFE liner caps #73048, open hole screw caps #73044, orequivalent; Clean 1 and 2 L glass bottles; Column—Vydac C4 214ATP54, aC4-silica reversed phase HPLC column with a dimension of 4.6×250 mm,particle size of 5μ and pore size of 300 Å; High-pressure liquidchromatography instrument capable of performing linear gradients (suchas Agilent 1100 HPLC equipped with a vacuum degasser, quaternary pump,thermostatted autosampler, thermostatted column compartment, diode arraydetector (DAD), and Chemstation chromatography software).

Reagents for this procedure included water (Milli-Q quality orequivalent) and solid chemicals are analytical grade or better andsolvents are HPLC grade or better, unless otherwise noted. The storageof reagents and procedural steps occurred at room temperarture, unlessotherwise indicated. Examples of such chemicals includedTRIS—Tromethamine, U.S.P. grade, Spectrum TR149, or equivalent;N-propanol, HPLC grade, 99.9%, Sigma Aldrich 34871, or equivalent;Ammonium Bicarbonate, Ultra>99.5%, Fluka #09830, or equivalent.

The Buffer for Deamidation Control was 30 mM Ammonium Bicarbonate, pH9.0. The Buffer for Oxidation Control was 50 mM TRIS, pH 7.5. Each ofthese solutions were sterile filtered using 0.22 μm PES filters (Corning#431098, or equivalent). Mobile phase: 710 ml 50 mM TRIS-HCl pH 7.5; 290ml n-propanol (or other appropriate volume with 71% 50 mM Tris-HCl, pH7.5 and 29% n-propanol). 6.05 g Tromethamine (USP grade, Spectrum#TR149, or equivalent) was dissolved in 0.95 L Milli-Q H₂O. The solutionwas brought to pH 7.5 with HCl and the volume brought up to 1 L withMilli-Q H₂O. After the mixing of the two solvents (TRIS and propanol),the mixture was sterile filtered using 0.22 μm PES filters (Corning#431098, or equivalent). The Conditioning Solution was 50% AcCN:H₂O,0.1% TFA.

Samples used as standards included World Health, Organization (WHO) rhGH(Cat. #98/574) reconstituted to 1.9-2.1 mg/ml with 1.0 ml of water and arhGH reference standard at 1.9-2.1 mg/ml concentration. The DeamidationResolution Solution was made by buffer exchanging the WHO standard into30 mM Ammonium Bicarbonate, pH 9.0 buffer using a PD-10, Nap-10, orNap-5 desalting column (depending on sample volume). The standard wasconcentrated using a 0.5 mL Vivaspin concentrator to 1.9-2.1 mg/ml, andthe sample was incubated at 37° C. for 24 hours. For the OxidationResolution Solution, the WHO standard was buffer exchanged into 50 mMTRIS, pH 7.5 buffer using a PD-10, Nap-10, or Nap-5 desalting column(depending on sample volume). The standard was concentrated using a 0.5mL Vivaspin concentrator to 1.9-2.1 mg/ml and H₂O₂ added to a finalconcentration of 0.015%. The reaction was incubated at 4° C. for 24 hrs.The reaction was stopped by adding 0.5-1 μl if 20 mg/ml catalase. Forthe test sample, the test material was diluted to 2.0 mg/ml proteinconcentration.

Procedure

The instrument was set-up with the following conditions: I) Column:Vydac C4 214ATP54 column; 2) Pump Setup—gradient: isocratic; flow rate:0.5 m/min; duration: 60 min; Max Pressure: 200 bar; 3) AutosamplerTemperature: 4° C.; 4) Injector Setup—Injection: standard Injection;Injection Volume: 20 μl; Draw Speed: 100 μl/min; Needle Wash: 100 ulwith water; Injection Speed: 100 μl/min; Stop Time: As pump; 5) DADSignals (Table 7);

TABLE 7 Sample Bw Reference Bw Units 220 4 600 100 nm 276 4 600 100 nm214 8 600 100 nm 220 4 600 100 nmPeak Width: >0.1 min; Slit: 4 nm, Stop Time: 60 min; 6) ColumnThermostat: Temperature: 45° C.; record the temperature; 7) PreliminaryIntegration Events (Chemstation Software, Agilent): Slope Sensitivity:0.1; Peak Width: 0.5; Area Reject: 1.0; Height Reject: 1.0; IntegrationON 10 min.

The column was pre-conditioned with 300 mL of conditioning solution (50%AcCN, H₂O, 0.1% TFA) at a flow rate between 0.5 and 1.5 ml/min.Pre-equilibration should be performed before a column has been used, orif peaks are broadening, re-condition the column with the conditioningsolution (200-300 mL). The column was equilibrated with 10 columnvolumes (41.5 ml=83 min at 0.5 ml/min) of the mobile phase.

20 μl of the standard was injected using the autosampler, and the HPLCprogram was run. If the retention time of the WHO standard was notbetween 32.5-35 min, the mobile phase composition was adjusted, thecolumn re-equilibrated, and the standard was re-run. Suggestedadjustments included adding less than 5 ml of 50 mM Tris-HCl pH 7.5 perliter of mobile phase if retention time is less than 32.5 min, and lessthan 2 ml of n-propanol if retention time greater than 35. Sinceevaporation of the propanol may occur, a standard was run each daysamples were to be tested and buffers were adjusted accordingly.

20 μl of the deamidation resolution solution was injected, and the HPLCprogram was run. Desamido-hGH appears as a small peak at a retentiontime of about 0.88±0.03 relative to the principal peak. The resolutionbetween the peaks corresponding to hGH and desamido-hGH was at least 1.0(current conditions result in a resolution of 1.29±0.04) and thesymmetry factor of the hGH peak is 0.8 to 1.8 (current conditions resultin a resolution of 1.26±0.06).

20 μl of the oxidation resolution solution was injected, and the HPLCprogram was run. Oxidized-hGH appears as a small peak at a retentiontime of about 0.8 relative to the principal peak.

20 μl of the test article was injected, and the HPLC program was run.Samples were run in triplicate. Average retention times were noted.

Data Analysis

The average retention time of the test article was compared with therhGH reference standard or the WHO standard. The average purity of thetest article was calculated: (Integration area of the mainpeak/integration areas of all peaks)×100%. Any peak(s) due to thesolvent were disregarded. Chromatograms showed absorbance (220 nm).

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons of ordinary skill in theart and are to be included within the spirit and purview of thisapplication and scope of the appended claims. All publications, patents,and patent applications cited herein are hereby incorporated byreference herein in their entirety for all purposes.

TABLE 8 SEQUENCES CITED. SEQ ID # Sequence Name 1 Full-length amino acidsequence of hGH 2 The mature amino acid sequence of hGH (isoform 1) 3The 20-kDa hGH variant in which residues 32-46 of hGH are deleted

1. A process of producing water soluble polymer-human growth hormone(hGH) polypeptide conjugate with a serum half-life at least two-foldgreater than the serum half-life of a naturally occurring, unconjugatedversion of an hGH protein, comprising: a) culturing recombinant hostcells capable of expressing an hGH polypeptide comprising anon-naturally encoded amino acid having the structure:

wherein the R group is any substituent other than one used in the twentynatural amino acids in a liquid nutrient medium containing thenon-naturally encoded amino acid under conditions which favor growth; b)expressing the hGH polypeptide at a concentration of at least 500 mg/L;c) eluting; d) collecting; e) substantially purifying said hGHpolypeptide from said cells or culture medium by conjugating saidnon-naturally encoded amino acid in said hGH polypeptide to a watersoluble polymer, wherein said non-naturally encoded amino acid issubstituted at a position selected from the group consisting of aminoacid positions 35, 92, 131, 134, 143, and 145 or any combination thereoffrom SEQ ID NO: 2 or the corresponding amino acids of SEQ ID NO: 1 or 3,f) contacting the conjugated hGH with an anion exchange chromatographymatrix under conditions that allow binding of the hGH polypeptide to thematrix g) eluting the matrix from e) and collecting the water solublepolymer-human growth hormone (hGH) polypeptide conjugate.
 2. The processof claim 1, wherein the water soluble polymer is polyethylene glycol. 3.The process of claim 1 further comprising h) contacting the hGHconjugate from step g) with a hydrophobic interaction chromatography(HIC) matrix under conditions that allow binding of the hGH to thematrix; and i) eluting and collecting the hGH conjugate from the HICmatrix to provide substantially pure hGH conjugate comprising anon-naturally encoded amino acid.
 4. The process of claim 1, whereinsaid recombinant host cell is a eukaryotic cell.
 5. The process of claim1, wherein said recombinant host cell is a prokaryotic cell.